Coupling agents for peptide synthesis

ABSTRACT

The present invention is directed to compounds of the formula (I) and (II) or salts thereof or N-oxides and their use in peptide synthesis.

This application claims priority benefit from International applicationno. PCT/US2004/036428, filed Nov. 1, 2004, and prior provisional patentapplication No. 60/516,167, filed Oct. 31, 2003, each of which isincorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

This work has been supported by a grant form the National Institutes ofHealth GM-09706 and the National Science Foundation (CHE-9003192). TheGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a new process for effecting theacylation step in amide formation, especially in peptide synthesis.

2. Description of the Prior Art

Polypeptides, especially proteins, play a critical role in fundamentalbiochemical processes in living cells. Biochemical reactions, includingmetabolic reactions, are catalyzed by enzymes, which are comprised ofproteins. These proteins are chiral molecules, and it is often the casethat of the various stereoisomers that may possibly exist, one isusually the most efficacious.

Moreover, polypeptides are useful as medicaments. In recent years,peptides have been found useful in combating various diseases, includingcancer, diabetes, plant toxins, and the like. Additionally, peptideshave shown specific activity as growth promoters, suppressants,antibiotics, insecticides, contraceptives, anti-hypertensives, sleepinducers, anti-depressants, analgesics, and so on.

The synthesis of proteins has always been a challenge to chemists.However, chemical synthesis offers advantages not realized by geneticengineering and other biological approaches such as isolation of naturalproteins. First, it is useful in confirming the structure of a protein.Moreover, protein synthesis is necessary to synthesize analogs, allowingscientists to evaluate biological activity and/or pharmacologicalefficacy in relation to molecular structure.

Success in the chemical synthesis of peptides relies, in part, on theuse of the appropriate coupling reagents in combination with theappropriate protecting groups. Especially in peptide synthesis,formation of the peptide bond between two amino acids requiresactivation of the carboxyl group of one of the amino acids before thereaction can occur. However, the activation step in conjunction with thecoupling reaction causes a serious problem of loss of configuration atthe carboxyl residue which has been activated. Thus, in designingchemical syntheses of peptides, the objective is to provide the peptideproduct in good yield and maintenance of the configurational integrityof the carboxylic component, i.e., minimal racemization. Thus, theduality of good yield and minimal or no racemization is difficult toachieve because the best methods require the acid to be converted to aderivative bearing a good leaving group. Thus, under normal couplingconditions, there is a loss of configuration.

Moreover, current methods of syntheses also tend to produce sidereactions which decrease yield.

Currently, syntheses of peptides are in solution by classical or variousrepetitive methods. Alternatively, peptides may be prepared on a solidsupport (Merrifield method). These are all popular techniques insynthesizing peptides from the coupling of two or more amino acids, insynthesizing larger peptides from the coupling of amino acids withsmaller peptides or in the coupling of smaller peptides. Solutionmethods have the advantage of being easily monitored, allowingpurification of intermediates, if necessary, at any stage. A majordrawback, however, is the relative slow pace of synthesis, with eachstep being carried out manually.

The major advantage of the Merrifield method is its easy automation sothat unattended, computer-controlled machine synthesis is possible.Unfortunately, the method suffers from an inherent deficiency due to theinsoluble nature of the support on which the synthesis proceeds. Unlesseach acylation step occurs with approximately 100% efficiency, mixtureswill inevitably be built up on the polymer. The longer the chain, thegreater will be the contamination by undesired side reactions. Sideproducts produced in such reactions remain to contaminate the desiredproduct when it is removed from the polymeric matrix at the end of thecycle. These current techniques are not useful in preparing peptides ofgreater than 40-50 residues; separation of side products from thedesired product becomes increasingly difficult when larger peptides aresynthesized.

For very long segments (50 or more amino acids), therefore, currentmethods are not satisfactory. Often, mixtures are obtained of suchforbidding complexity that it may be difficult or impossible to isolatethe desired peptide.

The problems enumerated hereinabove may be eliminated if the properderivatives of the underlying amino acids and/or the proper conditionsfor the coupling reaction could be found. Protecting groups, such ast-butyloxycarbonyl (t-Boc) or N-α-(9-fluorenylmethyl)oxycarbonyl (Fmoc),have been used to minimize side reactions.

The most commonly used coupling reagents are carbodiimides such asdicyclohexylcarbodiimides, diisopropylcarbodiimides,1-ethyl-3-(3′-dimethylaminopropyl)carbodiimides used with variousadditives.

Additives generally inhibit side reactions and reduce racemization.Heretofore, the most common peptide coupling additive used duringpeptide coupling for peptide synthesis is 1-hydroxybenzotriazole (HOBt).This reagent has been used either in combination with a carbodiimide orother coupling agent or built into a stand alone reagent, such as1-benzotriazolyoxytris (dimethylamino)phosphonium hexafluorophosphate(BOP) or an analogous uronium salt. HOBt is applicable to both stepwiseand segment condensations. However, many cases have been encountered inwhich HOBt is ineffective, possibly because of steric effects, or lowbasicity of the amino component. Especially problematic are segmentcouplings at amino acid units other than glycine or proline, since theproblem of racemization may be severe. The relatedN-hydroxybenzotriazinone (HOOBt) may provide better protection againstracemization, but it is rarely used due to competing side reactionsinvolving ring openings. A drawback in the use of BOP is that itproduces a toxic side product, hexamethylphosphorotriamide.

Recently other coupling reagents have been introduced, such asN-[1H-benzotriazo-1-yl)(dimethylamino)methylene]-N-methylmethanaminiumhexafluorophosphateN-oxide (HBTU),N-[(1-H-benzotriazol)dimethylamino)methylene]N-methylmethanaminiumtetrafluoroborate N-oxide (TBTU),O-(benzotriazol-1-yl)-1,3-dimethyl-1,3-dimethylene uraniumhexafluorophosphate (HBMDU),O-(benzotriazol-1-yl)-1,1,3,3-bis(tetramethylene)uroniumhexafluorophosphate (HBPyU) andO-(benzotriazol-1-yl)-1,1,3,3-bis(pentamethylene)uroniumhexafluorophosphate (HBPipU).

Another additive that has been used in peptide synthesis is3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine (HODhbt). HODhbt hasproved to be generally superior to HOBt. Moreover, its use permits oneto follow the completion of the reaction visually by a color changewhich occurs when acylation is complete. However, HODhbt has problemsassociated therewith due to inherent side reactions.

Other derivatives, which includeO-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate,O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-1,1,3,1-tetra-methyluroniumtetrafluoroborate and[3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)oxy]tris(pyrrolidino)phosphoniumhexafluorophosphate also have applications in peptide coupling.

Other reagents for facilitating peptide coupling have also beendescribed. For example, in Tetrahedron Letters, 1994, 2279-2282,Carpino, et al. disclose that 1-hydroxy-7-azabenzotriazole and itscorresponding uronium and phosphonium salts, designated HAPyU and AOP,respectively, were effective in promoting peptide coupling and avoidingracemization in a model solid-phase peptide segment coupling process. Inaddition, Carpino, et al. disclose in J. Org. Chem., 1994, 59, 695-698that azabenzotriazolyluronium salts, e.g., designated as HBTU, HATU,HAPyU, and HAMDU, are useful in peptide synthesis.

U.S. Pat. No. 5,644,029 to Carpino discloses, among other things, theuse of compounds of the following formula in promoting peptide coupling:

or N-oxides thereof or salts thereof wherein

R₁ and R₂ taken together with the carbon atoms to which they areattached form a heteroaryl ring wherein said heteroaryl ring is anoxygen, sulfur or nitrogen containing heteroaromatic containing from 3and up to a total of 13 ring carbon atoms, said heteroaryl may beunsubstituted or substituted with lower alkyl or an electron-donatinggroup;

Y is O, NR₄, CR₄R₅;

R₅ is independently hydrogen or lower alkyl;

X is CR₆R₇ or NR₆;

R₆ and R₇ are independently hydrogen or lower alkyl; or R₆ and R₇ takentogether form an oxo group or when n=0, R₄ and R₆ taken together mayform a bond between the nitrogen or carbon atom of Y and the nitrogen orcarbon atom of X;

Q is (CR₈R₉) or (NR₈);

when n is 1, R₄ and R₈ taken together may form a bond between the ringcarbon or nitrogen atom of Q and the ring carbon or nitrogen atom of R₈;

n is 0 or 1;

R₃ is hydrogen, lower alkyl carbonyl, aryl carbonyl, lower aryl alkylcarbonyl,

a positively charged electron withdrawing group, SO₂R₁₄, or

R₁₄ is lower alkyl, aryl or lower arylalkyl; q is 0-3;

R₈ and R₉ are independently hydrogen or lower alkyl or R₇ and R₈ takentogether with the carbon to which they are attached form an aryl ring,AA₁ is an amino acid and BLK is an amino protecting group, and m is 0 or1.

The present inventor has found other coupling agents, which providerelatively pure products with little, if any, side products beingco-produced and minimal, if any, racemization. Moreover, the reactionconditions are very mild and the reagents used are easy to prepare.Thus, by using the compounds of the present invention as additives, theyield of the peptide s enhanced and little, if any, racemization occurs.

SUMMARY OF THE INVENTION

The present invention relates to a compound of Formula I and the usethereof in the preparation of a peptide bond in peptide synthesis, saidcompound having the formula:

or N-oxide thereof or salts of said compounds of Formula I wherein

R₁ and R₂ taken together with the carbon atom to which they are attachedform an aryl or a heteroaryl ring, wherein said aryl ring is an aromaticring containing 6-14 ring carbon atoms and heteroaryl ring is an oxygen,sulfur, or nitrogen containing heteroaromatic containing at least 1 andup to 4 ring heteroatoms selected form oxygen, nitrogen and sulfur andcontaining from 3 and up to a total of 13 ring carbon atoms, said aryland heteroaryl may each independently be unsubstituted or substitutedwith lower alkyl or electron donating group or electron withdrawinggroup;

Q is CR₈R₉ or NR₈;

Y is O, NR₄ or CR₄R₅;

X is CR₆R₇ or NR₆;

R₅ is hydrogen or lower alkyl;

R₄ is hydrogen or lower alkyl or

R₄ and R₆ may form a bond between X and Y, when Y is NR₄ or CR₄R₅ andwhen Q is not present, or R₄ and R₈ may form a bond between Y and Q whenY is NR₄ or CR₄R₅ and Q is present, or R₆ and R₈ may form a bond betweenQ and X when Q is present;

R₈ and R₉ are independently hydrogen or lower alkyl, or when Q ispresent, R₈ taken together with R₄ may form a bond between Q and Y, whenY is NR₄ or CR₄R₅ or when Q is present, R₈ and R₆ may form a bondbetween Q and X;

R₆ and R₇ are independently hydrogen or lower alkyl or R₆ and R₇ takentogether form an oxo; or R₆, taken together with R₄, may form a bondbetween Y and X when Q is not present and Y is NR₄ or CR₄R₅ or R₆ and R₈taken together may form a bond between Q and X when Q is present; but inno circumstances is there a double bond between X and Q and Q and Y atthe same time;

n is O or 1;

R₃ is

R₁₀ is OR₁₂, lower alkyl, aryl, aryl lower alkyl, lower cycloalkyl,lower cycloalkyl lower alkyl, heterocyclic, heterocyclic lower alkyl,cycloalkenyl, or cycloalkenyl lower alkyl;

R₁₁ is OR₁₃, lower alkyl, aryl, aryl lower alkyl, lower cycloalkyl,lower cycloalkyl lower alkyl, heterocyclic or heterocyclic lower alkylcycloalkenyl or cycloalkenyl lower alkyl or R₁₀ and R₁₁ may be connectedby a bridging group consisting of S, O, NR₃₀ or (CHR₃₀)_(m), wherein R₃₀is lower alkyl or H and m is 1-3 such that R₁₀ and R₁₁ taken togetherwith the phosphorous atoms form a 5 or 6 membered ring;

R₁₂ and R₁₃ are independently lower alkyl, aryl, aryl lower alkyl, lowercycloalkyl, lower cycloalkyl lower alkyl, heterocyclic, heterocycliclower alkyl, cycloalkenyl, or cycloalkenyl lower alkyl;

Rings A₁ and B are independently aromatic rings containing 6 to 14 ringcarbon atoms or cycloalkenyl or cycloalkyl each containing 5 to 14 ringcarbon atoms;

T is CHR₃₁, O, S, or, NR₃₁;

R₃₁ is lower alkyl or H; and

Rb₁, Rb₂, Rc₁ and Rc₂ are independently hydrogen, lower alkyl or anelectron donating group.

In another embodiment, the present invention is directed to a compoundof formula II or to a salt, in which the cationic portion has thestructure of Formula II and to the use of the compound or salt:

or N-oxides thereof or salts of said compound of Formula II wherein

R₁ and R₂ taken together with the carbon atom to which they are attachedform a heteroaryl ring, wherein said heteroaryl is an heteroaromaticcontaining at least 1 and up to 4 ring heteroatoms selected from O, Sand N and containing from 3 and up to a total of 13 ring carbon atoms,said heteroaryl may be unsubstituted or substituted with lower alkyl orelectron donating group or electron withdrawing group;

Y₁ is N or CR₁₅;

R₁₅ is H or lower alkyl;

Q₁ is N or CR₁₆;

R₁₆ is H or lower alkyl; and

R₁₄ is hydrogen, a positively charged electron withdrawing group,SO₂R₁₇, lower alkyl carbonyl, aryl carbonyl, lower arylalkyl carbonyl,BLK₁ AA₁,

R₁₀ is OR₁₂, lower alkyl, aryl, aryl lower alkyl, lower cycloalkyl,lower cycloalkyl lower alkyl, heterocyclic, heterocyclic lower alkyl,cycloalkenyl or cycloalkenyl lower alkyl;

R₁₁ is OR₁₃, lower alkyl, aryl, aryl lower alkyl, lower cycloalkyl,lower cycloalkyl lower alkyl, heterocyclic, heterocyclic lower alkyl,cycloalkenyl or cycloalkenyl lower alkyl; or

R₁₀ and R₁₁ may optionally be connected by a bridging group T₁consisting of O, NR₃₀ or (CHR₃₀)_(m) wherein R₃₀ is lower alkyl or H andm is 1-3;

R₁₂ and R₁₃ are independently lower alkyl, aryl, aryl lower alkyl, lowercycloalkyl, lower cycloalkyl lower alkyl, heterocyclic, heterocycliclower alkyl, lower cycloalkenyl or cycloalkenyl lower alkyl;

rings A₁ and B are independently aromatic rings containing 6 to 14 ringcarbon atoms or cycloalkenyl or cycloalkyl, each containing 5 to 14 ringcarbon atoms;

T is O, S, NR₃₁ or CHR₃₁;

R₃₁ is H or lower alkyl; and

Rb₁, Rc₁, Rb₂ and Rc₂ are independently hydrogen, lower alkyl orelectron donating group;

R₁₇ is aryl, lower alkyl or lower arylalkyl, AA₁ is an amino acid orpeptide less a hydrogen atom on the N-terminus and an OH group on theC-terminus, and BLK₁ is an amino protecting group. In addition, thepresent invention is directed to the use of the compounds of Formula IIor, when R₁₄ is a positively charged electron withdrawing group, itssalt in which the cationic position has the structure of Formula II, inthe preparation of peptide.

An additional embodiment of the present invention is directed to aprocess for preparing a peptide bond from the reaction between an aminocompound and an acylating derivative of a carboxylic acid, said aminocompound being an amino acid or peptide and said carboxylic acid beingan N-terminal amino protected amino acid or an N-terminal aminoprotected peptide, which comprises reacting said amino compound and saidacylating derivative of a carboxylic acid in the presence of aneffective amount of a compound of formula I or formula II or to a salt,the cationic portion of which has the structure of Formula II, underconditions effective to form a peptide bond.

In another embodiment, the present invention is directed to a processfor forming an amide from the reaction of an organic amine having a freeamino and an acylating derivative of a carboxylic acid, which comprisesreacting said amine with the acylating derivative of the carboxylic acidwith an effective amount of a compound of Formula I or II or, when R₁₄is a positively charged electron withdrawing group, a salt, in which thecation has the structure of Formula II under amide forming conditions.

An additional embodiment of the present invention is directed to theprocess for synthesizing peptides comprising (a) reacting a firstNα-amino protected amino acid with a peptide synthesis resin underconditions effective to covalently link the amino acid to the resin, (b)cleaving the protecting group from the amino acid to form an amine witha free amino group, (c) reacting said amine with a second N α-aminoprotected amino acid in the presence of a peptide forming effectiveamount of a compound of Formula I or II or when R₁₄ us a positivelycharged electron withdrawing group, to a salt in which the cation hasthe structure of Formula II, said reaction being effected underpeptide-forming conditions, (d) repeating steps (b) and (c) until thedesired peptide is obtained and (e) removing the peptide from the resin.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

As used herein, the term “salt of Formula II” or any reference to a saltof Formula II refers to a salt of Formula II which consists of an anionand cation, the cation portion of which is positively charged. Thepositive charge may result from protonation, such as in the presence ofan acid of the compound or Formula II or it may result from R₁₄ being apositively charged electron withdrawing group. If reference is to bemade to a salt of Formula II in which the structure of Formula IIcontains a positively charged electron withdrawing group, the term“cation of Formula II” or its equivalent will be used.

As described hereinabove, an embodiment of the present invention relatesto compounds of Formula I or Formula II or to salts thereof or N-oxidethereof or cation of Formula I and their use in peptide coupling. Inother words, a first amino acid or a first peptide, each having a freeamino group is coupled with an acylating derivative of either a secondamino acid or a second peptide in the presence of compounds of Formula Ior II or salts thereof or N-oxides thereof or cation of Formula II underamide forming conditions to form a peptide bond and thus form a largerpeptide.

As employed herein, the term “heteroaryl” is a heteroaromatic containingat least one heteroatom ring atom selected from nitrogen, sulfur andoxygen and up to a maximum of four ring heteroatoms. The heteroarylcontains from 5 to 14 ring atoms and up to a total of 13 ring carbonatoms and a total of 18 carbon atoms. The heteroaryl group may bemonocyclic, bicyclic or tricyclic, although it is preferred that theheteroaryl is bicyclic and especially monocyclic. Also included in thisexpression are the benzoheterocyclics. The heteroaryl group preferablycontains no more than two ring heteroatoms, and most preferably containsone ring heteroatom. The most preferred ring heteroatoms are oxygen andnitrogen, with nitrogen being the most preferred.

If nitrogen is a ring atom, N-oxides can also be formed. The presentinvention contemplates the N-oxides of the nitrogen containingheteroaryls.

Examples of heteroaryls include thienyl, benzothienyl, 1-napthothienyl,thianthrenyl, furyl, benzofuryl, pyrrolyl, imidazolyl, pyrazolyl,pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, isoindolyl,indazolyl, purinyl, isoquinolyl, quinolyl, napthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, carbolinyl, isothiazolyl,isothiazolyl and the like. It is preferred that the heteroaryl group ispyridyl, pyrrolyl, furyl, indolyl, quinolyl, isoquinolyl or benzofuryl.Especially preferred is pyridyl.

When R₁ and R₂ taken together with the carbons to which they areattached form a tricyclic heteroaryl group, then the compounds ofFormula I or II is tetracyclic; if a bicyclic heteroaryl group is formedfrom R₁ and R₂ taken together with the carbons to which they areattached, then the compounds of Formula I or II are tricyclic. Finally,if R₁ and R₂ taken together form a monocyclic heteroaryl group, then thecompounds of Formula I or II are bicyclic. It is preferred thatcompounds of Formula I and II are tricyclic, and especially bicyclic.

The term “heterocyclic”, as used herein, when used alone or incombination with other groups, refers to a heterocyclic ring containingat least one heteroatom ring atom selected form nitrogen, sulfur andoxygen up to a maximum of 4 ring heteroatoms and from 5 to 14 ring atomsand up to a total of 18 carbon atoms. The heterocyclic group may bemonocyclic, bicyclic or tricyclic. It may be completely saturated or itmay be partially unsaturated, i.e., it may contain one or more doublebonds between ring atoms. It is preferred that the heterocyclic groupcontains 0, 1, 2, 3 or 4 double bonds. The term heterocyclic alsoincludes heteroaryl, as defined herein. Moreover, it is preferred thatthe heterocyclic moiety contains no more than two ring heteroatoms andmost preferably no more than one ring heteroatom. Examples includetetrahydrofuran, morpholinyl, piperazinyl, 2-tetrahydro quinolyl,3-tetrahydroquinolyl, 6-tetrahydroquinolyl or 7-tetrahydroquinolyl andthe like.

The term “lower alkyl”, when used alone or in combination with othergroups, refers to a carbon chain containing from one to six carbonatoms. It may be a straight chain or branched and includes such groupsas methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl,t-butyl, n-pentyl, amyl, hexyl and the like. The preferred lower alkylgroup contains from 1-3 carbon atoms, and is most preferably methyl.

The term “aryl” as used herein, alone or in combination, refers to anaromatic ring containing from 6-10 ring carbon atoms and up to a totalof 15 carbon atoms. It includes such groups as phenyl, α-naphthyl,β-naphthyl and the like. The preferred aryl is phenyl. It excludesheteroaryls.

Aralkyl groups are aryl groups attached to the main chain through analkylene bridge. Such groups include benzyl, phenethyl and the like.

“Lower alkyl carbonyl” refers to a lower alkyl group attached to themain chain through a carbonyl. Similarly, “aryl carbonyl” refers to anaryl group attached to the main chain through a carbonyl group.

“Lower cycloalkyl”, as used herein refers to a cycloalkyl groupcontaining 3-10 carbon ring atoms and up to a total of 15 carbon atoms.The cycloalkyl group may be monocyclic or bicyclic or tricyclic.Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, norbornyl, adamanyl, decalinyl, and the like.The preferred lower cycloalkyl groups are cyclopentyl and cyclohexyl.

“Lower cycloalkyl lower alkyl” is a lower cycloalkyl group attached tothe main chain through an alkylene bridge. Such groups includecyclohexylmethyl, cyclopentylethyl and the like.

“Cycloalkenyl” refers to a lower cycloalkyl group, as defined herein,containing at least one double bond and up to a maximum of 6carbon-carbon double bonds. It is not completely aromatic; but it mayinclude an aromatic moiety. It may contain one ring or two or more ringsfused together. The double bonds may be located in one ring or bothrings. One or more rings may be completely aromatic, while the remainingrings, if any, in the structure may each be completely saturated orcontain 1 or 2 double bonds. It is to be noted however, thatcycloalkenyl, as described herein, excludes aryl. Examples includecyclohexenyl, cyclooctenyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- or 8-indanyl, andthe like.

As used herein, an “electron donating group” shall designate a groupthat will release or donate electrons more than hydrogen would if itoccupied the same position in the molecule. See., J. March, AdvancedOrganic Chemistry, 3^(rd) Ed., John Wiley & Sons p. 237 (1985). Thesetypes of groups are well known in the art. Examples include loweralkylamino, diloweralkylamino, amino, halo, aryl, lower alkoxy, loweraralkoxy, aryloxy, mercapto, lower alkylthio, and the like. Thepreferred electron donating groups are amino, hydroxy, lower alkoxy,lower alkylamino and diloweralkylamino.

The term “electron withdrawing groups” as defined herein refers to agroup that will draw electrons to itself more than a hydrogen atom wouldif it occupied the same position in the molecule. See., J. March,Advanced Organic Chemistry, 3^(rd) Ed., John Wiley & Sons p. 237 (1985).They include such groups as nitro, monohaloalkyl, dihaloalkyl,trihaloalkyl (e.g., CF₃), halo, formyl, lower alkanoyl, loweralkylsulfonyl, lower alkylsulfinyl, carboxy, lower alkoxy carbonyl,sulfonamido, amido, CONR₅₀R₅₁, wherein R₅₀, and R₅₁ are independentlyhydrogen lower alkyl, aryl, aryl lower alkyl, heterocyclic; heterocycliclower alkyl, lower cycloalkyl, lower cycloalkyl lower cycloalkyl loweralkyl, cycloalkenyl cycloalkenyl lower alkyl and the like.

A “positively charged electron withdrawing group” is an electronwithdrawing group bearing a positive charge and forming a stable bond toa N-hydroxide (N—O). These types of groups are well known in the art.Examples include uronium groups,

e.g.,

imino cations, e.g.,

or phosphonium cations, e.g.,

and the like, wherein R₁₈, R₁₉, R₂₀, R₂₁, R₂₂, R₂₃, R₂₄ and R₂₅ areindependently hydrogen, lower alkyl, lower alkoxy lower alkyl or if theimino cation is formed, R₁₈ ad R₂₀ taken together with the nitrogenatoms to which they are attached and the carbon atom therebetween mayform a ring containing up to 6 ring atoms and up to a total of 5 ringcarbon atoms or R₁₈ and R₁₉ taken together with the nitrogen atom towhich they are attached or R₂₀ and R₂₁ taken together with the carbonatoms to which they are attached may form a 5 or 6-membered heterocyclicring containing up to a total of 5 ring carbon atoms or if the uroniumcation is formed, R₂₀ and R₁₈ may be taken with the nitrogen to whichthey are attached form a 5 or 6 membered heterocyclic ring containing upto a total of 5 ring carbon atoms or both R₁₈ and R₁₉ taken togetherwith the nitrogen atoms to which they are attached or R₂₀ and R₂₁ takentogether with the nitrogen atom to which they are attached, may eachsimultaneously form a 5 or 6-membered heterocyclic ring, each ringcontaining up to a total of 5 ring carbon atoms. In the uronium andimino cations, it is preferred that R₁₈ and R₁₉ and R₂₀ and R₂₁, whenpresent, are the same. It is especially preferred that R₁₈, R₁₉, R₂₀,R₂₁, whenever present, are the same. It is also more preferred that therings formed with respect to the uronium, imino, and phosphonium cationsare 5 or 6 membered rings.

With respect to the phosphonium cation, R₁₈ and R₁₉ and/or R₂₀ and R₂₁and/or R₂₂ and R₂₃ may, each be independently taken together with thenitrogen atoms to which they are attached to form a ring. Thus, thephosphonium cation may be comprised of 1 ring, two rings or three rings.It is preferred that R₁₈ and R₁₉, or R₂₀ and R₂₁ or R₂₂ and R₂₃ are thesame. It is especially preferred that R₁₈, R₁₉, R₂₀, R₂₁, R₂₂ and R₂₃are the same.

With respect to cations of Formula II, preferred cyclic uronium andimino groups have the formula

wherein R₁₉ and R₂₀ are as defined hereinabove and n₁ and n₂ areindependently 0 or 1, and U₁ and U₂ are independently O, CH₂ or NH orN-Alk wherein Alk is lower alkyl.

In the above formulae, the preferred values of R₁₈, R₁₉, R₂₀ R₂₁, R₂₂and R₂₃ are independently methyl, ethyl, n-butyl, pentyl andCH₂CH₂—O—CH₂CH₃. It is preferred that R₁₈, R₁₉, R₂₀ (for all) and R₂₁,when present (for imino), and R₂₂ and R₂₃ (for phosphonium) whenpresent, are the same.

The preferred values of R₁₈, R₁₉, R₂₀, R₂₁, when present, and R₂₂, andwhen present, and R₂₃, when present, are independently lower alkyl,especially methyl. It is preferred R₁₈, R₁₉, R₂₀ and R₂₁, when present,R₂₂, when present, and R₂₃, when present, are all the same. Further itis preferred that all are methyl.

With respect to the uronium and/or imino cations, when R₁₈ and R₁₉ orR₂₀ and R₂₁ taken together form a ring, they may form heterocyclicmoieties of the formula:

whereinU=CH₂, O, NH or N-Alk, wherein Alk is lower alkyl, especially methyl.

Preferred cyclic groups present in the phosphonium cations also have theformula

wherein U, is defined hereinabove.

It is preferred that R₁ and R₂ taken together with the carbon atoms towhich they are attached form an aryl or heteroaryl ring wherein the arylring is a phenyl or naphthyl and the heteroaryl ring contains 5 to 10ring atoms and one or two ring heteroatoms consisting of O, S, N and 3-8ring carbon atoms.

With respect to compounds of Formula I, the following are preferred.When n is 1, it is preferred that R₇ and R₈ are hydrogen or lower alkyl,but most preferably hydrogen.

Preferred values of Y are S, O, NR₄, or CR₄R₅, wherein R₄ and R₅ areindependently hydrogen or methyl. Especially preferred values of Y areO, CH₂ and NH. It is also preferred, however, that Q is absent and Yforms a double bond with X.

It is preferred that X is CR₆R₇ or NR₆. Preferred values of R₆ and R₇are hydrogen or lower alkyl.

With respect to compounds of Formula I, it is preferred that Q is notpresent and Y and X are independently N or CH. It is more preferred thatat least one of Y and X is N and the other is CH. It is even morepreferred that Y is N and X is CH or N or that Y and X are both N.

When R₆ and R₇ taken together form an oxo group, X becomes C═O. It ismost preferred that X is C═O, CH₂ or NH or N(CH₃). Moreover, anembodiment of Formula I has the formula

In cases when n is 0, then R₄ and R₆ taken together may form a bondbetween X and Y, i.e., a bond may form between the ring carbon atom of Xand the ring carbon atom of Y, or between the ring nitrogen atom of Xand the ring nitrogen atom of Y, or the ring nitrogen atom of X, and thering carbon atom of Y or the ring carbon atom of X and the ring nitrogenatom of Y. In other words, under these circumstances when n is O and R₄and R₆ taken together form a bond between X and Y, the compound ofFormula I becomes

wherein R₁, R₂, Y, X, and R₃ are as defined above. Under thesecircumstances, it is preferred that Y is CH or N and X is CH or N. It ismost preferred that Y and X are N.

When n is 1, the compound of Formula I becomes

wherein

R₁, R₂, Q, R₈, R₉, Y, R₄, R₅, X, R₆, R₇ and R₃, R₁₀, R₁₁, R₁₂ and R₁₃are as defined above. It is preferred that R₈ and R₉ are hydrogen.

As indicated hereinabove, when n is 1, R₄ and R₈ taken together may forma bond between Q and Y, i.e., the ring carbon atom of R₄ and the ringcarbon atom of R₈ may form a bond, or the ring carbon atom of R₄ and thering nitrogen atom of R₈ may form a bond, or the ring nitrogen atom ofR₄ and the ring carbon atom of R₈ may form a bond or the ring nitrogenatom of R₄ and the ring nitrogen atom of R₈ may form a bond. Forexample, under these circumstances, the compound of Formula I becomes:

The preferred values of X in this formulations are C═O or NH or CH₂.

When n is 1, preferred values of Q are CH₂ or NH. However, it is alsopreferred that the compounds of Formula I have the formula:

wherein

Q is CR₉ or N, and

R₉ is hydrogen or lower alkyl

and R₁, R₂, X, OR₃ and Y are as defined hereinabove. Examples ofcompounds of Formula I include:

wherein R₁, R₂ and R₃ are as defined hereinabove.

It is also preferred that compounds of Formula I have the formula:

wherein R₁, R₂, R₃, Y and X are as defined hereinabove.

The most preferred compounds of Formula I have the formula:

or N-oxides thereofwherein Q, Y, X, R₃, n, are as defined hereinabove,

A is N or CR₂₄;

D is CR₂₅ or N;

E is CR₂₆ or N;

G is CR₂₇ or N; and

R₂₄, R₂₅, R₂₆ and R₂₇ are independently hydrogen or lower alkyl or anelectron donating group or R₂₅ and R₂₆ or R₂₄ and R₂₅ or R₂₆ and R₂₇taken together form with the carbon atom to which they are respectivelyattached an aryl ring. It is preferred that A, D, E or G all are CH andmore preferably at least one of A, D, E, G is N.

It is preferred that no more than two of A, D, E, G are N. It is mostpreferred that only one of A, D, E, G is N. Further it is preferred thatR₂₄, R₂₅, R₂₆ or R₂₇ are hydrogen or an electron-donating group, asdefined herein, The preferred electron donating group is lowerdialkylamino especially N,N-dimethyl amino and lower alkoxy, e.g.methoxy.

Preferred compounds of Formula III have the formulae:

or N-oxides thereofwherein Y, X, n, Q and R₃ are as defined hereinabove and R₂₄ and R₂₅ areindependently lower alkyl, hydrogen or an electron donating group.

Of the compounds of Formula IV-VII, when n is 1, the most preferredcompound is that of Formula IVa

or N-oxides thereofwherein Q, Y, X and R₃ are as defined hereinabove and R₂₄ and R₂₅ arelower alkyl or hydrogen or an electron donating group.

Preferred compounds of Formula I also have the formula

or N-oxides thereofwherein n, Q, D, E, X and Y are as defined hereinabove and J is NR₂₈, O,CR₂₈R₂₉ or S(O)p, and p is 0, 1, 2.

R₂₈ is hydrogen, lower alkyl or electron donating group as definedhereinabove and R₂₉ is hydrogen or lower alkyl. It is preferred that R₂₉is hydrogen. The preferred values of R₂₈ is an electron donating groupor hydrogen.

Preferred values of J are O or S(O)p; the preferred value of p is 1.

Preferred compounds of Formula VIII when n is 1 have the formula:

or N-oxides thereofwherein J, Y, R₈, R₉, n and R₃ are as defined hereinabove and X is C═O.

In compounds VIII, IX, and VIIIa as depicted above, at least one of D,E, or J is a heteroatom. Furthermore, it is most preferred that at mosttwo of J, E, and D are heteroatoms. It is most preferred that only oneof J, E, and D is a heteroatom.

When n is O, preferred compounds of Formula I becomes:

Thus, the present invention includes compounds having the formula:

or N-oxides thereof.wherein A, D, E, G, Y, X, R₃ and J are as defined hereinabove.

The compounds of Formula I more preferably are compounds of the formula:

or N-oxides thereof.

In the above formulae, when the ring contains Y═X, this means that R₄ ofY and R₆ of X are joined together to form a ring bond between the Y ringatom and the X ring atom, so that as depicted hereinabove there is adouble bond between the Y ring atom and the X ring atom.

Furthermore, in the above formulae, when the ring contains Y═N, then R₄of Y and R₈ of NR₈ of Q join together to form a ring bond so that thereis a double bond between the nitrogen ring atom and the Y atom. Thus, Yis CR₅ or N under these circumstances.

The most preferred embodiment of Formula I has the formula:

wherein one or two of A, D, E, G, is N and the rest are CH and X is CHor N. It is most preferred that X is N. It is also preferred that atmost one of A, D, E and G is N and the rest are CH. It is most preferredthat A is N and especially G is N.

Preferred embodiments of compounds of Formula I include:

or the N-oxides thereof.

With respect to compounds of Formula I, R₃ is as defined hereinabove.The various groups on R₁₀, R₁₁, R₁₂ and R₁₃, e.g., alkyl, aryl,arylalkyl, cycloalkyl, cycloalkylalkyl, heterocyclic, heterocyclic loweralkyl or lower cycloalkyl heterocyclics may be unsubstituted, orsubstituted by lower alkyl or electron donating or electron withdrawinggroups. It is preferred that the groups are unsubstituted or substitutedby lower alkyl.

With respect to compounds of Formula I, it is preferred that R₁₀ isOR₁₂, lower alkyl, aryl or aryl lower alkyl. It is more preferred thatR₁₀ is OR₁₂ or aryl.

It is also preferred that R₁₁ is OR₁₃, lower alkyl, aryl or aryl loweralkyl. It is most preferred that R₁₁ is OR₁₃ or aryl. Preferred valuesof R₁₂ and R₁₃ each independently are lower alkyl, aryl or aryl or aryllower alkyl. It is most preferred that R₁₂ and R₁₃ are alkyl having 1-3carbon atoms, or phenyl. It is also preferred that R₁₂ and R₁₃ are thesame.

It is also preferred that R₁₀ and R₁₁ are connected to each other by abridging group, T₁. Preferred values of T₁ are O, CH₂, S, or NR₃₀ whenR₃₀ is lower alkyl and more preferably H. When R₁₀ and R₁₁ are joinedtogether, then R₃ becomes

wherein R₁₀, R₁₁ and T, are as defined hereinabove. As defined herein R₃may be defined as

It is preferred that rings A₁ and B are independently aromatic,especially phenyl.

It is preferred that R₃ is

wherein R₁₀′ and R₁₁′, R₁₂ and R₁₃ are independently lower alkyl, arylor aryl lower alkyl and Rb₁, Rc₁, Rb₂ and Rc₂ are independently hydrogenor lower alkyl. It is also preferred that R₁₂ and R₁₃ are connected by abridging group T₁ to form the following R₃ moiety

In an embodiment of the present invention, R₁₀ and R₁₁ are connected bythe bridging group T₁, as depicted hereinabove. Preferred values of T₁are CH₂, O, S and NH and most preferably CH₂ and O.

Embodiments of R₃ include

wherein R₁₆ and R₁₇ are independently lower alkyl, R_(b1) and R_(c1) areindependently H or lower alkyl and T is as defined hereinabove andpreferably O, NH or CH₂.

Examples of R₃ include

Of course, various combinations and permutations of the formulaedescribed herein are also contemplated by the present invention. Inaddition, Markush groupings containing less than all of the elementsdescribed hereinabove as well as the various permutations thereof arealso contemplated by the present invention.

The compounds of Formula I can be prepared by art recognized techniques.An illustrative technique is described hereinbelow.

For example, compounds of formula

can be prepared by reacting

under substitution reaction conditions wherein R₁, R₂, Y, Q, n, R₃, R₁₀and R₁₁ are as defined hereinabove and L is a leaving group, such ashalo and the like.

It is preferable that the reaction is run in an inert polar organicsolvent and that the reactants are soluble therein at room temperature.It is also preferred that the product of the substitution is insolublein the solvent at room temperature. Examples of suitable solvents forthe reaction include chloroform, carbon tetrachloride, ethyl ether,dioxane, tetrahydrofuran and methylene dichloride, and the like. Thereaction takes place at effective temperatures, which may range from themelting point of the solvent to reflux temperature thereof but it ispreferred that the reaction take place at about room temperature or atslightly elevated temperatures up to the reflux temperature of thesolvent. It is especially preferred that the reaction take place at roomtemperature or at slightly elevated temperatures, such as up to 60° C.

Compounds of Formula 100 can be prepared as described in U.S. Pat. No.RE 37,686, RE 38,073, 5,580,981, 5,644,029, 5,698,675, the contents ofwhich are incorporated by reference.

For example, compounds of Formula 100, such as

can be prepared by reacting hydrazine with

wherein R′ is halogen, NH—NH₂ or OR″, and

R″ is lower alkyl, such as methyl. This reaction is performed atslightly elevated temperatures, such as 70-100° C., although thereaction may be performed at temperatures ranging from room temperatureto the boiling point of the solvent.

The reaction is usually run in an organic solvent in which the reactantsare insoluble at room temperature, but in which the reactants andproduct are soluble at slightly elevated temperatures. Examples ofuseful solvents include ethanol, DMF and the like. In many cases, thereis a color change in the reaction mixture, indicating the formation ofthe product. Work-up, such as removal of the solvent, followed byacidification provides the desired product.

The hydrazino derivative (R′═NH—NH₂) can be prepared by reacting thecorresponding halide, such as chloride or bromide, with hydrazine undersubstitution reaction conditions. The ether derivative (R¹═OR″) can beprepare by reacting the corresponding alcohol with an alkylatingreagent, such as Me₂SO₄/Na₂CO₃, under ether forming conditions.

Compounds of Formulae 100a, 100b, or 100c are useful for preparingcompounds of Formula I. These latter compounds can also be prepared byart-recognized techniques. For example, compounds of Formula I areprepared by reacting compounds of 100a, 100b, 100c, respectively, with

under substitution reaction conditions wherein R₁₀ and R₁₁ are definedhereinabove and L is a leaving group, such as halo, (e.g., Cl, Br, I)and the like.

The N-oxides can be prepared from the compounds of Formula I having anitrogen ring heteroatom in the heteroaryl group. These N-oxides areprepared by art-recognized techniques such as by oxidation thereof, suchas with peracid; e.g., peracetic acid or m-chloroperbenzoic acid.

With respect to compounds of Formula II, it is preferred that Y₁ is N orCR₁₅, wherein R₁₅ is hydrogen or methyl. Especially preferred values ofY₁ are CH and N.

It is also preferred that Q₁ is N or CR₁₆ wherein R₁₆ is hydrogen orlower alkyl. The preferred value of Q₁ is N or CH.

With respect to compounds of Formula II, preferred values of Y₁ and Q₁are CH or N. In a preferred embodiment, the compound of Formula II hasthe formula

wherein R₁ and R₂ and R₁₄ are as defined as hereinabove and Q₁ is N orCH, but especially N.

Another embodiment of the compound having Formula II or salt of FormulaII, wherein the cation has the formula is:

wherein

R₁ and R₂ taken together with the carbon atoms to which they areattached form an heteroaryl ring wherein said heteroaryl ring is anoxygen, sulfur or nitrogen heteroaromatic containing from 3 to 13 ringcarbon atoms and 1-4 heteroatoms selected from O, S and N, saidheteroaryl ring may be unsubstituted or substituted with lower alkyl orelectron donating group;

Y₁ is N or CR₁₅;

R₁₅ is H or lower alkyl;

Q₁ is N or CR₁₆;

R₁₆ is H or lower alkyl;

R₁₄ is hydrogen, a positively charged electron withdrawing group,

SO₂R₁₇, lower alkyl carbonyl, aryl carbonyl, lower alkyl aryl, orBLK₁-AA₁

R₁₇ is aryl, aryl lower alkyl or lower arylalkyl;

AA₁ is an amino acid or peptide less a hydrogen atom on the N-terminusand an OH on the C-terminal;

BLK₁ is an amino protecting group,

R₁₀ is OR₁₂, lower alkyl, aryl, aryl lower alkyl, lower cycloalkyl,lower cycloalkyl lower alkyl, heterocyclic, heterocyclic lower alkyl,lower cycloalkenyl, lower cycloalkenyl or lower cycloalkenyl loweralkyl;

R₁₁ is OR₁₃, lower alkyl, aryl, aryl lower alkyl, lower cycloalkylheterocyclic, heterocyclic lower alkyl, lower cycloalkenyl, lowercycloalkenyl lower alkyl;

and R₁₀ and R₁₁ may optionally be connected by a bridging group selectedfrom the group consisting of O, S, NR₃₀, or (CHR₃₀)_(m), wherein eachR₃₀ is independently lower alkyl or hydrogen and m is 1-3;

R₁₂ and R₁₃ are independently lower alkyl, lower cycloalkyl, lowercycloalkyl lower alkyl, heterocyclic, heterocyclic lower alkyl, lowercycloalkenyl, or lower cycloalkenyl lower alkyl;

ring A₁ and ring B are independently aromatic containing 6 to 14 ringcarbon atoms or cycloalkenyl or cycloalkyl, each containing 5 to 14 ringcarbon atoms;

R_(b1), R_(c1), R_(b2), R_(c2) are independently hydrogen, lower alkylor electron donating group;

T is CHR₃₁, O, S or NR₃₀; and

R₃₁ is hydrogen or lower alkyl.

Another embodiment of the compound having Formula II or salt of FormulaII, wherein the cation has the formula is:

or N-oxide or salt thereofwherein one of Y₁ and Q₁ is CR₁₅ and the other is N or CH;

R₁₅ is H or lower alkyl;

R₁ and R₂ taken together with the carbon atom to which they are attachedform an aryl or heteroaryl ring wherein said aryl ring is an aromaticring containing 6-14 ring carbon atoms and said heteroaryl ring is anoxygen, sulfur or nitrogen heteroaromatic containing from 3 to 13 ringcarbon atoms and 1-4 heteroatoms selected from O, S and N, saidheteroaryl ring may be unsubstituted or substituted with lower alkyl orelectron donating group;

R₁₄ is hydrogen, a positively charged electron withdrawing group,

SO₂R₁₇, lower alkyl carbonyl, aryl carbonyl, lower alkyl aryl, orBLK₁-AA₁

R₁₇ is aryl, aryl lower alkyl or lower arylalkyl;

R₁₁ is OR₁₃, lower alkyl, aryl, aryl lower alkyl, lower cycloalkylheterocyclic, heterocyclic lower alkyl, lower cycloalkenyl, lowercycloalkenyl lower alkyl;

R₁₀ is OR₁₂, lower alkyl, aryl, aryl lower alkyl, lower cycloalkyl,lower cycloalkyl lower alkyl, heterocyclic, heterocyclic lower alkyl,lower cycloalkenyl, lower cycloalkenyl or lower cycloalkenyl loweralkyl;

and R₁₀ and R₁₁ may optionally be connected by a bridging group selectedfrom the group consisting of O, S, NR₃₀, or (CHR₃₀)_(m), wherein eachR₃₀ is independently lower alkyl or hydrogen and m is 1-3;

AA₁ is an amino acid or peptide less a hydrogen atom on the N-terminusand an OH on the C-terminal;

BLK₁ is an amino protecting group,

R₁₂ and R₁₃ are independently lower alkyl, lower cycloalkyl, lowercycloalkyl lower alkyl, heterocyclic, heterocyclic lower alkyl, lowercycloalkenyl, or lower cycloalkenyl lower alkyl;

ring A₁ and ring B are independently aromatic containing 6 to 14 ringcarbon atoms or cycloalkenyl or cycloalkyl, each containing 5 to 14 ringcarbon atoms;

R_(b1), R_(c1), R_(b2), R_(c2) are independently hydrogen, lower alkylor electron donating group;

T is CHR₃₁, O, S or NR₃₁; and

R₃₁ is hydrogen or lower alkyl.

As defined herein, in some embodiments of Formula II, R₁ and R₂ takentogether with the carbon atoms to which they are attached form an arylring. It is preferred that the aryl ring is naphthyl and is especiallyphenyl.

Moreover, in Formula II, as defined herein R₁₄ is preferably H or apositively charged electron withdrawing group, as defined hereinabove,or

wherein R₁₀ and R₁₁ are as defined herein and the electron withdrawinggroup is as defined hereinabove. It is most preferred that R₁₄ is

The preferred values of R₁₀ and R₁₁ are as defined hereinabove withrespect to Formula I.

Preferred structures of Formula II have the formula:

wherein

Y₁ is N or CR₁₅;

R₁₅ is H or lower alkyl;

Q₁ is N or CR₁₆;

R₁₄ is hydrogen, a positively charged electron withdrawing group,

R₁₆ is H or lower alkyl;

SO₂R₁₇, lower alkyl carbonyl, aryl carbonyl, lower alkyl aryl orBLK₁-AA₁; AA₁ is an amino acid or peptide less a hydrogen atom on theN-terminus and an OH on the C-terminus;

BLK₁ is an amino protecting group;

R₁₀ is OR₁₂, lower alkyl, aryl, aryl lower alkyl, lower cycloalkyl,lower cycloalkyl lower alkyl, heterocyclic, heterocyclic lower alkyl,lower cycloalkenyl, or lower cycloalkenyl lower alkyl;

R₁₁ is OR₁₃, lower alkyl, aryl, aryl lower alkyl, lower cycloalkyl,lower cycloalkyl lower alkyl, heterocyclic, heterocyclic lower alkyl,lower cycloalkenyl or lower cycloalkenyl lower alkyl;

and R₁₀ and R₁₁ may optionally be connected by a bridging group selectedfrom the group consisting of O, S, NR₃₀, or (CHR₃₀)_(m), wherein eachR₃₀ is independently lower alkyl or hydrogen and m is 1-3; and

R₁₂ and R₁₃ are independently lower alkyl, lower cycloalkyl, lowercycloalkyl lower alkyl, heterocyclic, heterocyclic lower alkyl, lowercycloalkenyl or lower cycloalkenyl lower alkyl;

ring A₁ and ring B are independently aromatic containing 6 to 14 ringcarbon atoms or cycloalkenyl or cycloalkyl each containing 5 to 14 ringcarbon atoms, and

R_(b1), R_(c1), R_(b2), R_(c2) are independently hydrogen, lower alkylor electron donating group and T is CHR₃₁, O, S or NR₃₁ wherein R₃₁ ishydrogen or lower alkyl;

A is N or CR₂₄;

D is N or CR₂₅;

E is N or CR₂₆;

G is CR₂₇ or N;

R₂₄, R₂₅, R₂₆ and R₂₇ are independently hydrogen or lower alkyl or anelectron donating group or R₂₅ and R₂₆ or R₂₄ and R₂₅ or R₂₆ and R₂₇taken together with the carbon atoms to which they are attached form anaryl ring, but at least one of A, D, E and G is N.

It is also preferred that structures of Formula II have the formula:

wherein

Y₁, Q₁ and R₁₄, are as defined hereinabove,

D is CR₂₅ or N;

G is CR₂₆ or N;

J is NR₂₈, O, CR₂₈R₂₉ or S(O)_(p);

R₂₅ and R₂₆ are independently hydrogen or lower alkyl or an electrondonating group or R₂₅ and R₂₆ taken together with the carbon atoms towhich they are attached form an aryl group;

R₂₈ is hydrogen or electron donating group or lower alkyl;

R₂₉ is hydrogen or lower alkyl and

p is 0, 1 or 2.

More preferred structures of Formula II have the formula

wherein

A is N or CR₂₄,

D is CR₂₅ or N,

E is CR₂₆ or N.

G is CR₂₇ or N;

J is NR₂₈, O, CR₂₈, R₂₉ or S(O)_(p); and

R₂₄, R₂₅, R₂₆, R₂₇, R₂₈, R₂₉, p, Q₁, and R₁₄ are as defined hereinabove.

With respect to Formula II, and all of its various embodiments depictedhereinabove, it is preferred that no more than two of A, D, E, and G areN. It is most preferred that only one of A, D, E, or G is N. Further, itis preferred that R₂₄, R₂₅, R₂₆ or R₂₇ are independently hydrogen or anelectron donating group, as defined herein. The preferred electrondonating groups are lower dialkylamino, especially N,N-dimethylamino andlower alkoxy, e.g., methoxy.

Moreover, it is preferred that T is CH₂, O, S or NH and more preferablyCH₂ or O. Preferred structures of Formula X have the formulae

or N-oxides thereof, wherein R₂₄, R₂₅, Q₁ and R₁₄ are as definedhereinabove.

Of the structures of Formulae XI-XIV, the most preferred compound isthat of XIa viz.

wherein R₂₄, R₂₅, Q₁ and OR₁₄ are as defined herein.

Preferred structures of Formula II also have the formula

wherein J, E, D, Q₁ and R₁₄ are as defined hereinabove. It is preferredthat R₂₄ is hydrogen, lower alkyl or electron donating group as definedhereinabove and R₂₅ is hydrogen or lower alkyl. It is most preferredthat R₂₅ is hydrogen and it is most preferred that R₂₄ is an electrondonating group or hydrogen.

Preferred values of J are O or S(O)p or NH; the preferred value of p is1.

Preferred structures of Formula II also have the following formula:

or the N-oxides thereof.

In the various structures described herein whether it is for compoundsof Formula I or II or any other embodiment of the present inventiondepicted herein, the preferred values of T is O, S, NH or CH₂. Inaddition, in the various structures depicted hereinabove, it ispreferred that m is 1 and that R₃₀ is H.

Compounds of Formula II or salts, especially wherein the cationicportion has the structure of Formula II, are prepared by art recognizedtechniques. For example compounds of Formula II are prepared by reactingcompounds of Formula XVII whenever R₁₄ is hydrogen

with R₁₄L, wherein R₁, R₂, Q₁, and R₁₄ is as defined herein and L is aleaving group, such as halo, (e.g. chloro, bromo or iodo). However, ifR₁₄ is a positively charged electron withdrawing group, then thestructures of Formula II is a cation, and in this case, there would bean anion associated with this cationic moiety. For example, when R₁₄ is

the compounds of Formula XVII is reacted with

under substitution reaction conditions wherein R₁₂ and R₁₁ and L are asdefined hereinabove. It is preferable that reaction is run in an inertpolar organic solvent and that the reactants are soluble therein at roomtemperature. It is also preferred that the product is insoluble in thesolvent at room temperature Examples of the solvent that could be usedinclude chloroform, carbon tetrachloride, ethyl ether, dioxane,tetrahydrofuran, methylene, chloride, and the like. The reaction takesplace at effective temperatures, which may range from the melting pointof the solvent up to reflux temperatures, but it is preferred that thereaction takes place at about room temperature or at slightly highertemperatures up to the reflux temperature of the solvent. It isespecially preferred that the reaction take place at room temperature orat slightly elevated temperatures such as up to 60° C.

Compounds of Formula XVII can also be prepared by art recognizedtechniques known to one of ordinary skill in the art. An exemplaryprocedure is as follows:

For example, a compound of Formula VII is refluxed with acetic anhydrideto give the corresponding anhydride under anhydride formationconditions. The anhydride product was then treated with acetamide underamide forming conditions to give the corresponding cyclic imide XIX. Thecyclic imide XIX is subjected to conditions effective for it to undergoHoffman rearrangement, e.g., by reacting it with sodium hypobromite (orsodium hydroxide and bromine) followed by hydrolysis. For example, thecyclic imide XIX is reacted with NaOBr, Copper (II) acetate and H₂S toprovide the amino carboxylic acid (XX) with one less carbon atom.Esterification of acid XX under the esterfying conditions gives thecorresponding ester XXI. Treatment of XXI with hydroxylamine in acid(hydroxylammonium salt) under esterification reaction conditions givesthe hydroxamic acid derivative XXII. Diazotization followed byintramolecular cyclization gives the azo derivatives XXIII. On the otherhand, reaction of the hydroxamic with formic acid under effectiveconditions, such as by heating the hydroxamic acid with formic acid ateffective temperatures e.g., temperatures ranging from just above roomtemperature up to and including reflux temperatures, and preferably, atthe reflux temperature of formic acid gives the product XXIV.

Of course, various combinations and permutations of the formulaedescribed herein are also contemplated by the present invention. Inaddition, Markush groupings containing less than all of the elementsdescribed hereinabove as well as the various permutations thereof arealso contemplated by the present invention.

As described herein, the compounds or salts or N-oxides describedhereinabove are useful in promoting peptide coupling, i.e., the reactionbetween a free amino group of a first amino acid or first peptide with afree carboxy group or acylating group of a second amino acid or peptide.The process of the present invention is general; it can be used ineffecting the coupling of a dipeptide of an amino acid, a tripeptide andan amino acid, dipeptides, pentapeptide, higher peptides, polypeptides,etc.

When the compound of Formula I or structures of Formula II reacts withan amino compound such as an amino blocked amino acid or protein of theformula BLK₁-AA₁, the corresponding amino acid ester of the one of thefollowing compounds is formed depending on the identity of the couplingagent:

wherein AA₁ is an amino acid or protein as defined herein, BLK₁ is ablocking group as defined herein and Y, Q, Q₁, n, X, R₁, and R₂ are asdefined hereinabove. This amino acid ester can then react with acompound having a free amino group such as an arylamino, alkylamino,lower aryl amino, heterocyclic amino, heterocyclic lower alkylamino,lower cycloalkylamino, lower cycloalkyl lower alkyl amino, and the likedesignated as R₃₃R₃₄ NH, wherein R₃₃ and R₃₄ are independently hydrogen,lower alkyl, aryl or lower aryl alkyl, to form a compound of theformula:

Removal of the blocking group by techniques known to one skilled in theart affords the product:AA₁NR₃₃R₃₄.

This technique is extremely useful when the second amino compound is anamino acid or peptide having a free amine group, designated as AA₂. Forexample, if the coupling agent is a compound of Formula I, a peptide maybe formed between AA₁ and AA₂ as follows, for example,

wherein AA₁, AA₂, BLK₁, R₁, R, Y, Q, n and X are as defined herein.

If the coupling agent contains a structure of Formula II,

whether compound or cationic portion of the salt, then the reactionbecomes

In the reaction AA₁, AA₂, BLK₁, R₁, R₂, and Q₁ are as defined herein.

The blocking group can be any of the blocking groups known in the artbut the preferred blocking groups are FMOC, BOC, benzyloxycarbonyl BSMOCand Bspoc.

The term “amino acid” or AA, AA₁, or AA₂ as used herein refers to anorganic acid containing both a basic amino group (NH₂) and an acidiccarboxyl group. (COOH).

Therefore, said molecule is amphoteric and exists in aqueous solution asdipole ions. (See “The Condensed Chemical Dictionary”, 10^(th) Ed.,edited by Gessner G. Hawley, Van Nostrand Reinhold Company, London,England p. 48 (1981). The preferred amino acids are the α-amino acids.They include but are not limited to the 25 amino acids that have beenestablished as protein constituents. They must contain at least onecarboxyl group and one primary or secondary amino group in the aminoacid molecule. The term includes such proteinogenic amino acids asalanine, valine, leucine, isoleucine, norleucine, proline,hydroxyproline, phenylalanine, tryptophan, 2,4-diamino butyric acid,methionine, glycine, serine, threonine, cysteine, cystine, glutamicacid, lysine, hydroxylysine, ornithine, arginine, histidine,penicillamine, naphthylamine, α-phenylglycine, aspartic acid,asparagines, glutamine, arginine, tyrosine, and the like.

As used herein, the term “peptide” refers to the class of compoundscomposed of amino acid units chemically bound together with amidelinkages. A peptide may contain as little as two amino acid residues ormay consist of a polymer of amino acid residues (polypeptide).

As used herein, the terms “amino acid” and “peptide” also include aminoacids and peptides, respectively containing blocking (protecting)groups. These protecting “groups” block the amino group or the carboxylgroup of the amino acid or peptide not involved in or taking part in thecoupling in order to prevent unwanted side reactions. These protectinggroups also protect reactive groups on the side chain.

A number of blocking reagents for amino groups are known in the art andhave been utilized in the syntheses of peptides. These blocking groupsare discussed in U.S. Pat. Nos. 3,835,175, 4,508,657, 3,839,396,4,581,167, 4,394,519, 4,460,501 and 4,108,846, the contents of all ofwhich are incorporated by reference as if fully set forth herein. Otheramino protecting groups are discussed in U.S. Pat. Nos. 5,221,754,5,510,491 and 5,637,719 the contents of which are also incorporated byreference. Other amino protecting groups are described in an articleentitled “Solid Phase Peptide Synthesis”, by G. Barany and R. B.Merrifield in THE PEPTIDES, Vol. 2, edited by E. Gross and J.Meienhoffer, Academic Press, N.Y., N.Y. 100-118 (1980), and in the bookentitled “PROTECTIVE GROUPS IN ORGANIC SYNTHESIS” by T. W. Green, JohnWiley & Sons, New York, the contents of all of which are beingincorporated by reference.

The term amino acid protecting group, (BLK, BLK₁) as used herein, refersto blocking groups which are known in the art and which have beenutilized to block the amino NH₂) group of the amino acid. Blockinggroups such as 9-lower alkyl-9-fluorenyloxycarbony,2-chloro-1-indanylmethoxy-carbonyl (CLIMOC) andbenz[f]indene-3-methyloxycarbonyl (BIMOC) and dbd-TMOC are discussed inU.S. Pat. Nos. 3,835,175, 4,508,657, 3,839,396, 4,581,167, 4,394,519,4,460,501 and 4,108,846 referred to hereinabove, the contents of whichare incorporated by reference. Moreover, other amino protecting groupssuch as 2-(t-butyl sulfonyl)-2-propenyloxycarbonyl (Bspoc) andbenzothiophene sulfone-2-methoxycarbonyl (Bsmoc). Other N-aminoprotecting groups include such groups as the t-butyloxycarbonyl (BOC),t-amyloxycarbonyl (Aoc), β-trimethylsilyl-ethyloxycarbonyl (TEOC),adamantyl-oxycarbonyl (Adoc), 1-methyl-cyclobutyloxycarbonyl (Mcb),2-(p-biphenylyl)propyl-2-oxycarbonyl (Bpoc),2-(p-phenylazophenyl)propyl-2-oxycarbonyl (Azoc),2,2-dimethyl-3,5-dimethyloxybenzyloxycarbonyl (Ddz),2-phenylpropyl-2-oxycarbonyl (Poc), benzyloxycarbonyl (Cbz),p-toluenesulfonyl aminocarbonyl (Tac), o-nitrophenylsulfenyl (Nps),dithiasuccinoyl (Dts), Phthaloyl, piperidine-oxycarbonyl, formyl,trifluoroacetyl and the like.

These protecting groups can be placed into four categories:

-   -   1) a base labile Nα-amino acid protecting group such as FMOC,        and the like.    -   2) protecting groups removed by acid, such as Boc, TEOC, Aoc,        Adoc, Mcb, Bpoc, Azoc, Ddz, Poc, Cbz, 2-furanmethyloxycarbonyl        (Foc), p-methoxybenzyloxycarbonyl (Moz), Nps, and the like.    -   3) protecting groups removed by hydrogenation such as Dts, Cbz.    -   4) protecting groups removed by nucleophiles, such as Bspoc,        Bsmoc and Nps and the like.    -   5) protecting groups derived from carboxylic acids, such as        formyl, acetyl, trifluoroacetyl and the like, which are removed        by acid, base or nucleophiles.

A variety of carboxy protecting groups known in the art may be employed.Examples of many of these possible groups may be found in “ProtectiveGroups in Organic Synthesis”, by T. W. Green, John Wiley & Sons, 1981,the contents of which are incorporated by reference. These examplesinclude such groups as methyl ester, t-butyl ester,β-trimethylsilylethyl ester, benzyl ester and the like.

In addition, during the course of protein synthesis, it may be necessaryto protect certain side chains of the amino acids to prevent unwantedside reactions. The various protecting groups are discussed in U.S. Pat.No. 5,360,920, the contents of which are incorporated herein byreference.

The term “acylating group of an amino acid or peptide” refers to a groupon the free carboxy end of the amino acid or peptide that facilitatesthe acylation reaction, i.e., nucleophilic substitution at the acylcarbon. Examples include the free acid, acid halide, esters, such aslower alkyl esters, phenoxy esters which are unsubstituted orsubstituted with 1-5 electron withdrawing groups as defined herein; oran anhydride and the like. The preferred acylating derivative is theacid, acid halide, especially the acid chloride or fluoride, and thephenoxy ester.

The preferred acylating amino acid is an amino acid group of the formulaBLK-AA-M,wherein BLK is an amino protecting group

-   -   AA is an amino acid less the H on the COOH moiety and    -   M is halo or

wherein Ra is independently halo, lower alkyl, nitro, cyano or otherelectron withdrawing group and b is 0-5. When b is 0, the phenyl groupis unsubstituted.

The most preferred acylating group of an amino acid is the amino acidchloride or fluoride. The preparation and use of amino acid chlorides asan acylating derivative is discussed in an article by Carpino, et al. inJ. Org. Chem., 1986, 51, 3734-3736, the contents of which areincorporated herein by reference. Briefly, amino acid chlorides can beprepared by reacting the amino acid with thionyl chloride andrecrystallizing the product from a recrystallization reagent, such asCH₂Cl₂-hexane.

The preparation and use of amino acid fluorides in peptide synthesis arediscussed U.S. Pat. No. 5,360,920, the contents of which areincorporated herein by reference. As described therein, the amino acidfluorides can be prepared by reacting an N-protected amino acid with thereagent cyanuric fluoride. This reaction can be run at temperatures aslow as 0° C. and up to the refluxing temperature of the solvent, but itis preferred that the reaction is run at room temperature. It can alsobe run in an inert solvent, such as pyridine/CH₂Cl₂ and the like. Thecyanuric fluoride can be prepared from the corresponding chloride in thepresence of potassium fluoride at elevated temperatures ranging from150° to 250° C., according to the following equation

A typical preparation of the peptide in accordance with the presentinvention involves the following steps

1) protection of the free carboxyl group in a first amino acid or afirst peptide, unless the amino acid or peptide is anchored to a solidsupport.

2) protection of the free amino group of a second amino acid or peptide.

3) protection of the side chains, if necessary.

4) coupling the first amino acid or peptide with the second amino acidor peptide in the presence of compounds of Formula I.

5) removal of the protecting groups.

The procedure of steps 1-3 can be performed in any order.

In the coupling step, the compounds of Formula I or II or salts orN-oxides thereof or cation of Formula II is present in effectiveamounts. Usually, the first amino acid or peptide is present inapproximately equimolar amounts with the second amino acid or peptide.Furthermore, the amount of the compound having Formula I or II useddepends upon the amount of peptide or amino acid which is present in theleast amount (i.e. the limiting reagent); thus the molar ratio of thecompound of Formula I or II to the amino acid or peptide present in theleast molar amount, ranges from about 1:3 to about 3:1, although it ispreferred that approximately equimolar amounts of the compound ofFormula I or II (or salt or N-oxide thereof or cation of Formula II) thefirst amino acid or peptide and the second amino acid or peptide beused.

The coupling reaction usually takes place in an inert organic solventsuch as dimethylformamide (DMF) or ethers, such as ethyl ether, THF ordioxane. In fact DMF is the preferred solvent in the solid phasesynthesis because of its favorable solvation properties. The reactiontakes place under mild conditions usually ranging from about 0° C. toabout 30° C. After the peptide is formed, the blocking groups areremoved by techniques known to one skilled in the art.

The following sequence is illustrative of the coupling reaction; in theexamples below, amino acids (AA) are used, although the procedure isgeneral for amino acids and/or peptides:

In the above scheme, BLK is an amino acid blocking group, AA₁, AA₂ andAA₃ are first, second and third amino acid respectively and P is acarboxy protecting group.

As shown by the above scheme, the N-α amino protected amino acid isreacted with a second amino acid in which the carboxy group isprotected.

A peptide is formed between the first amino acid and the second aminoacid. The peptide chain can be increased by removing the alpha aminoprotecting group by techniques known to one skilled in the art and thenreacting the corresponding dipeptide with another N-t amino protectedamino acid in the presence of a compound of Formula I to form thecorresponding tri-peptide. The N-α amino protecting group of thetri-peptide is removed and the above cycle is repeated until the desiredpeptide has been obtained.

The present invention can readily be utilized in solid phase peptidesynthesis. Solid phase peptide synthesis is based on the stepwiseassembly of a peptide chain while it is attached at one end to a solidsupport or solid phase peptide resin. Two methods are generally wellknown in the art.

One, the Merrifield method, employs a solid support for attachment ofthe amino acid or peptide residues. This method employs N-protectedamino acids as building blocks which are added to an amino acid orpeptide residue attached to the solid support at the acyl (acid) end ofthe molecule. After the peptide bond has been formed, the protectinggroup is removed and the cycle repeated. When a peptide having thedesired sequence has been synthesized, it is then removed from thesupport.

The second method, the inverse Merrifield method, employs reagentsattached to solid supports in a series of columns. The amino acid orpeptide residue is passed through these columns in a series to form thedesired amino acid sequence.

These methods are well known in the art as discussed in U.S. Pat. Nos.4,108,846, 3,839,396, 3,835,175, 4,508,657, 4,623,484, 4,575,541,4,581,167, 4,394,519 as well as in Advances in Enzymology, 32, 221(1969) and in PEPTIDES, Vol. 2, edited by Erhard Gross and JohannesMeienhoffer, Academic Press, New York pp. 3-255 (1980) and the contentsthereof are incorporated herein by reference as if fully set forthherein.

The compounds of the present invention are useful as coupling agents orbases in peptide coupling. However, their effectiveness is also afunction of the solvent system which is used to dissolve the reactantsand bases used in the coupling reaction. If the compound of the presentinvention is not too soluble in the solvent used in the coupling method,then it should be converted to a form more soluble in the solvent or thesolvent should be changed to one in which it is soluble. This is usuallynot a problem in peptide coupling conducted in solution. But, with somepeptide synthesizers, the option of changing the solvent is notavailable. For example, with some synthesizers, DMF orN-methylpyrrolidone is the solvent utilized. However, compounds of thepresent invention may not be too soluble in the solvent utilized, suchas DMF or N-methylpyrrolidone. Thus, to overcome this problem thecompound of the present invention is converted to a compound which issoluble in the solvent utilized, such as DMF or N-methylpyrrolidone. Forexample, if the coupling agent or base of the present invention is thatof Formula I and if R₁₀ or R₁₁ is aryl or heteroaryl or if A₁ or B isaryl or heteroaryl, then the trick to overcome this problem is to placea t-butyl or t-amyl group or any other group wherein the carbon atomattached to the aryl or heteroaryl ring is a tertiary carbon.

If R₁₀ or R₁₁ or A₁ or B contain more than one ring it is preferred thatthe t-butyl or amyl group or other tertiary carbon group, such as atertiary hydrocarbyl group (containing only carbon and hydrogen atoms)is attached to the ring attached to the phosphorus atom. If more thanone such group is attached to the rings, it is preferred that thesegroups are the same.

The following examples further illustrate the present invention.

In the examples, the following abbreviations are utilized:

HOAt 1-hydroxy-7-azabenzotriazole DIEA diisopropylethylamine TEAtriethylamine CBZ or Cbz benzyloxycarbonyl EDCN-ethyl-N′-[3-dimethylaminopropyl] carbodiimide hydrochloride Aibaminoisobutyric acid THF tetrahydrofuran DMF dimethylformamide TCFH1,1,3,3-tetramethylchloroformamidinium hexafluorophosphate PyClu1,1,3,3-bis(tetramethylene)chlorouronium hexafluorophosphate PyBroptris(pyrrolindino)bromophosphonium hexafluorophosphate TFAtrifluoroacetic acid TMP 2,4,6-trimethylpyridine PCA p-chloroanilineDPOPOAt diphenoxyphosphoryloxy-7-azabenzotriazole HDATUO-(3,4-dihydro-4-oxo-5-azabenzo-1,3-diazin-3-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate N-HATU1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo- [4,5-b]pyridinhexafluorophosphate 3-oxide O-Dhad3-oxy-4-oxo-3,4-dihydro-5-azabenzo-1,2,3-triazine O-HATUN-[(1H-1,2,3-triazolo[4,5-b]pyridin-1-yloxy)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate HDTUO-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-y1)-1,1,3,3-tetramethyluronium hnexaflurorphosphate HODhat3-hydroxy-4-oxo-3,4-dihydro-5-azabenzo-1,2,3-triazine HODhbt3-hydroxy-4-oxo-3,4-dihydro-1,2,3-benzotriazine DEPOAtdiethoxyphosphoryloxy-7-azabenzotriazole DtpOAtdi(o-tolyl)phosphinyloxy-7-azabenzotriazole HODhad3-hydroxy-4-oxo-3,4-dihydro-5-azabenzo-1,3-diazine Odhad3-oxy-4-oxo-3,4-dihydro-5-azabenzo-1,3-diazine HDADUO-(3,4-dihydro-4-oxo-5-azabenzo-1,3-diazin-3-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate HDAPyUO-(3,4-dihydro-4-oxo-5-azabenzo-1,2,3-triazin-3-yl)-1,1,3,3-bis(tetramethylene)uronium hexafluorophosphate PyDAOP[(3,4-Dihydro-4-oxo-5-azabenzo-1,2,3-triazin-3-yl)oxy]tris(pyrrolidino)phosphonium Hexafluorophosphate DtpOBtdi(o-tolyl)phosphinyloxybenzotriazole DtpODhbt3-[di-o-tolyl)phosphinyloxy]-3,4-dihydro-4-oxo-2,3 benzotriazine DEPDBt3-(diethoxyphosphoryloxy)-3,4-dihydro-4-oxo-1,2,3- benzotriazine DPOPDBt3-(diphenoxyphosphoryloxy)-3,4-dihydro-4-oxo-1,2,3- benzotriazineDPOPOAt diphenoxyphosphoryloxy-7-azabenzotriazole PyDOP[(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)oxy]tris(pyrrolidino)-phosphonium hexafluorophosophate HDPyUO-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate DIC diisopropylcarbodiimide DCHMAN,N-dicyclohoxylmethylamine DmppOAt 2,8-dimethylphenoxaphosphinyloxy-7-azabenzotriazole Bt benzotriazole Pfppentafluorophenyl ester N-HBTU1-[bis(dimethylamino)methylene]-1H-benzotriazoliumhexafluorophosphate-3-oxide O-HBTUN-[1H-benzotriazol-1-yloxy)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate

EXAMPLE 1 Diethoxyphosphoryloxy-7-azabenzotriazole (DEPOAt)

A solution of HOAt (1.36 g, 10 mmol) and triethylamine (TEA) (1.39 mL,10 mmol) in 15 ml of dry benzene was cooled in an ice bath. To thesolution was added dropwise with stirring a solution of diethylchlorophosphate (1.72 g, 1.45 ml, 10 mmol) in 10 ml of dry benzene underN₂. The addition was completed in 10 min, and stirring was continued atice-bath temperature for 1 hour and then at room temperature for anadditional 4 hours. The reaction mixture was cooled to 5-10° C. and TEAHCl was removed quickly by filtration. The colorless clear solution wasevaporated to dryness while the temperature was kept below 35° C. Dryhexane was added to the oily residue and the whole was tightly cappedunder N₂, and placed in a refrigerator (−20° C.) for 2 hours. The oilyresidue solidified as a white solid, which was then recrystallized fromdry CH₂Cl₂-hexane to give 1.74 g (63%) of the ester as colorless plates:mp 48-50° C.; ¹H NMR (CDCl₃): δ 8.77 (dd, 1H), 8.40 (dd, 1H), 7.44 (dd,1H), 4.57 (m, 4H, 2CH₂), 1.47 (2t, 2CH₃); IR (film on NaCl plate): 2987(m), 1596 (m), 1395 (m), 1306 (s), 1026 (vs), 838 (m), 775 (s), 699 (m)cm⁻¹.

Anal. Calcd for C₉H₁₃N₄0₄P: C, 39.71; H, 4.81; N, 20.58.

Found: C, 39.84; H, 4.72; N, 20.54.

EXAMPLE 2 Di(o-tolyl)phosphinyloxy-7-azabenzotriazole (DtpOAt)

A solution of HOAt (0.136 g, 1 mmol) and DIEA (0.21 ml, 1.2 mmol) in 10ml of dry CH₂Cl₂ was cooled in an ice bath and 0.2647 g (1 mmol) ofdi(o-tolyl)phosphinoyl chloride (DTP-Cl) was added to the solutionportion wise with stirring under N₂. The stirring was continued in theice-bath for 30 minutes and then at room temperature for 5 hours. Theresulting colorless solution was washed with cold saturated NaHCO₃solution (2×10 ml) and brine (2×10 ml), and dried over MgSO₄ to give0.26 g (69%) of white solid after removing the solvent.Recrystallization from CH₂Cl₂-hexane gave 0.21 g of an analytically puresample of the ester as colorless block crystals: mp 170-172° C.; ¹H NMR(CDCl₃): δ 8.74 (dd, 1H), 8.32 (d, 1H), 7.99 (dd, 1H), 7.56 (m, 2H,Ar—H), 7.35 (m, 6H, Ar—H), 2.70 (d, 6H, 2CH₃); IR (KBr): 3064 (w), 1592(s), 1458 (m), 1379 (m), 1334 (m), 1238 (sh, s), 1113 (s), 928 (m), 811(s), 770 (s), 691 (s) cm¹.

Anal. Calcd for C₁₉H₁₇N₄O₂P: C, 62.62; H, 4.70; N, 15.37.

Found: C, 62.23; H, 4.72; N, 15.47.

EXAMPLE 3 Synthesis of3-hydroxy-4-oxo-3,4-dihydro-5-azabenzo-1,2,3-triazine (HODhat)

A. Quinolinimide. 2,3-Pyridinedicarboxylic acid (22.5 g, 0.135 mol) wasadded to 25 ml of acetic anhydride and the mixture was heated withstirring to reflux on a steam-bath. A clear colorless solution wasobtained after about 1 hour, and the heating was continued for 2 morehours. Then, acetic acid and excess acetic anhydride were distilled off(˜12.6 g) until the temperature of the residual solution reached 165° C.The residue which remained in the flask solidified to a dust red solidupon cooling to room temperature.

To the above solid, acetamide (14 g, 0.23 7 mol, 1.75 equiv) was addedand the mixture was heated overnight by means of an oil-bath, keepingthe temperature at 120-125° C. After cooling to room temperature, thesolid was collected and washed with acetic acid (2×10 ml) and water(3×50 ml). The gray solid was suspended in 250 ml of hot water and themixture stirred for 15 min to give by filtration 15 g (75%) of theabove-identified imide as a gray solid: mp 237°-239° C. (lit. mp 233°C.).

B. 3-Aminopicolinic Acid. Quinolinimide (50 g, 0.344 mol) was dissolvedin 1000 ml of 10% NaOH while cooling in an ice-bath and to the solutionwas added slowly with stirring and cooling a cold NaOBr solution, whichhad been obtained by mixing 56 g (18 ml, 0.344 mmol) of Br₂ with 350 mlof 15% NaOH in an ice-bath. After the addition had been completed,stirring was continued in the ice-bath for 15 min and at roomtemperature for 1 hour. The resulting mixture was then heated to 85° C.with stirring for 1 hour. After cooling, the mixture was neutralized topH 5-6 by means of 50% H₂SO₄ and the whole was kept at room temperatureovernight.

The resulting white cloudy mixture was heated and the clear solutiontreated with a hot solution of 22 g (0.11 mol) of copper (II) acetatemonohydrate in 400 ml of H₂O and 10 ml of acetic acid. The mixture washeated with a steam bath for 15 min and then cooled at room temperatureand the copper salt collected and washed twice with cold water.

The copper salt was re-suspended in 500 ml of water, and H₂S was passedthrough the suspension for 2 to 3 hours while stirring. Black CuS wasremoved from the mixture by filtration and the filtrate decolorized withcharcoal. Removal of water gave a dust yellow solid, which wasrecrystallized form water-ethanol (1:1 v/v) to give 29 g (60%) of theabove-identified acid as a cream-yellow solid: mp 212-214° C. (lit. mp210° C.).

C. Ethyl 3-Aminopicolinate. A mixture of 3-aminopicolinic acid (5.07 g,36 mmol), absolute ethanol (20 ml) and concentrated H₂SO₄ (6 ml) wasrefluxed for 48 hours. After cooling, the mixture was concentrated toabout 15 ml and poured into 15 g of ice. The mixture was basified withconcentrated aqueous ammonia to pH 8-9 with cooling in an ice bath, andthe white precipitate that separated was collected by filtration. Thefiltrate was extracted with ether (4×50 ml), and the ether layer washedwith brine (4×50 ml) and dried over MgSO₄. Evaporation of the ethersolution afforded another solid. Both fractions were combined andrecrystallized from benzene-hexane to give 4.05 g (68%) of theabove-identified ethyl ester as white needles: mp 126-127.5° C. (lit mp131-133° C., yield 42%); ¹H NMR (CDCl₃): δ8.09 (dd, 1H), 7.23 (dd, 1H),7.04 (dd, 1H), 5.76 (s, 2H, NH₂), 4.46 (q, 2H, CH₂), 1.45 (t, 3H, CH₃).

D. 3-Amino-2-picolinehydroxamic Acid. Hydroxylamine hydrochloride (16.3g, 0.233 mol) was added slowly with stirring and cooling to 1100 ml ofaqueous NaOH solution prepared from 18.7 g (0.467 mol) of NaOH. To thesolution, 19.4 g (0.116 mol) of the ethyl ester prepared in Section Chereinabove was added portion wise followed by 110 ml of methanol, andthe mixture was stirred at room temperature for 48 hours. The solutionwas concentrated under reduced pressure to about 100 ml and neutralizedwith cooling to pH 5-6 with 25% HCl. A white precipitate that separatedwas collected by filtration, washed with a small amount of cold water,and dried over P₂O₅ in vacuo to give 17.8 g (100%) of the3-amino-2-picoline-hydroxamic acid as a white solid, which was pureenough for the next step. An analytical sample of this acid was obtainedin 90% yield after two recrystallizations from MeNO₂-MeOH-EtOAc as whiteblock-like crystals: mp 131-133° C. (lit. mp 143-145° C., yield 49%); ¹HNMR (DMSO-d₆CDCl₃): δ10.91 (s, 1H, OH), 8.89 (s, 1H, NH), 7.74 (t, 1H),7.16 (d, 2H), 6.71 (s, 2H, NH₂); IR (KBr): 3443 (m), 3334 (s), 1660 (s,CON), 1606 (s), 1262 (w), 1017 (w), 805 (m) cm¹.

E. 3-Hydroxy-4-oxo-3,4-dihydro-5-azabenzo-1,2,3-triazine (HODhat). To asuspension of finely powdered product produced in step D hereinabove(7.3 g, 47.6 mmol) in 28 ml of water, 8.5 ml of concentrated HCl wasadded while stirring. While cooling in an ice-bath, a cold solution ofNaNO₂ (4.93 g, 71.4 mmol) in 5 ml of water was added dropwise to themixture and the temperature was maintained below 5° C. After completionof the addition, stirring in the ice-bath was continued for another 30minutes, and the solid which had precipitated was collected byfiltration, washed with a small amount of cold water and air dried togive 2.52 g (32%) of the triazine as a yellow solid: mp 195° C.(explodes) [lit mp: 195° C. (explodes)]. The analytical sample (2.1 g)was obtained by recrystallization from EtOHwater (9:1 v/v) as lightorange-yellow needles: mp 203° C. (explodes); ¹H NMIR (DMSOd₆): δ9.13(dd, 11H), 8.65 (dd, 111), 8.08 (dd, 11H); IR (KBr): 2600 (broad, OH),1713 (vs, C(O)N), 1574 (s), 1420 (m), 1230 (sh, s), 1185 (s), 1066 (sh,s), 974 (sh, m), 794 (m) cm⁻¹.

3-Hydroxypicolinic acid (1.6 g, 20%) was also isolated from the motherliquor as light pink needles: mp 222-224° C. (lit. mp 220-222° C.).Anal. Calcd for C₆H₅NO₃: C, 51.80; H, 3.62; N, 10.07. Found: C, 51.52;H, 3.58; N, 9.98.

EXAMPLE 4 3-Hydroxy-4-oxo-3,4-dihydro-5-azabenzo-1,3-diazine (HODhad)

A mixture of 1.224 g (8 mmol) of the product produced in Step D ofExample 3 and 3 ml of 98% formic acid were heated under reflux for 15minutes after which 8 ml of water was added, and the whole was boiledfor 15 minutes and cooled to room temperature. The precipitate thatseparated was collected by filtration and washed with water (2×5 ml).After being boiled with ethanol twice, there was obtained 0.71 g (55%)of HODhad, which was obtained in an analytically pure form as a yellowsolid: mp 318.5-320° C. (dec.); ¹H NMR (DMSO-d₆): δ12.15 (br, 1H, OH),8.85 (dd, 1H), 8.69 (s, 1H, CH), 8.17 (dd, 1H), 7.84 (dd, 1H); IR (KBr):2625 (broad, OH), 1683 (sh, vs, CON), 1600 (w), 1446 (m), 1410 (s), 1359(s), 1223 (s), 990 (s), 902 (m), 791 (s) cm⁻¹.

Anal. Calcd for C₇H₅N₃O₂: C, 51.53; H, 3.09; N, 25.76.

Found: C, 51.46; H, 3.00; N, 25.80.

EXAMPLE 5 CBZ-Aib-ODhat

In an ice bath, 0.3168 g of EDC.HCI (1.65 mmol) was added with stirringto a suspension of CBZ-Aib-OH (0.3 555 g, 1.5 mmol) and HODhat (theproduct of Ex. 4) (0.246 g, 1.5 mmol) in 10 ml of THF and 5 ml of DMF.The resulting mixture was stirred at room temperature for 3 hours.Solvents were removed in vacuo and the oily residue was extracted with40 ml of EtOAc. The EtOAc solution was washed with 5% aqueous citricacid (3×10 ml), 10% NaHCO₃ solution (3×10 ml) and brine (3×10 ml), anddried over MgSO₄.

Evaporation of solvent gave a cream yellow oily residue, whichsolidified after drying in vacuo over P₂0₅ overnight. The crude solidwas purified by flash chromatography with EtOAc as eluent to give 0.46 g(80%) of the above-identified ester as a cream yellow solid: mp 57-59°C.; ¹H NMR (CDCl₃): δ 9.05 (dd, IH), 8.49 (dd, IH), 7.86 (dd, IH), 7.26(m, 5H, C₆H₅), 5.47 (s, IH, NH), 5.10 (s, 2H, CH₂), 1.73 (s, 6H, 2CH₃);IR (KBr): 1813 (m, COO), 1735 (vs, NCO), 1267 (s), 1052 (s) cm⁻¹.

Anal. Calcd for C₁₈H₁₇N₃0₅: C, 56.39; H, 4.47; N, 18.27.

Found C, 56.10; H, 4.56; N, 18.08.

EXAMPLE 6 CBZ-Aib-ODhad

As described for EXAMPLE 6, 0.2447 g (1.5 mmol) of HODhad (the productof Ex. 5) was treated with 0.3555 g (1.5 mmol) of CBZ-Aib-OH in thepresence of EDC HCl in 15 ml of a THF-DMF mixture (2:1) to give 0.52 g(90%) of the above product as a white solid: mp 48-50° C.; ¹HNMR(CDCl₃): δ 8.89 (dd, 1H), 8.13 (dd, 1H), 7.92 (s, 1H, CH), 7.71 (dd,1H), 7.36 (m, 5H, C₆H₅), 5.47 (s, 1H, NH), 5.15 (s, 2H, CH₂), 1.75 (s,6H, 2CH₃); IR. (KBr): 1809 (s, COO), 1718 (vs, NCO), 1590 (sh, m), 1522(sh, m), 1468 (m), 1274 (sh, s), 1059 (s), 974 (w) cm⁻¹.

Anal. Calcd for C₁₉H₁₈N₄0₅: C, 59.68; H, 4.74; N, 14.65.

Found: C, 59.43; H, 4.80; N, 14.58.

EXAMPLE 7

Under an atmosphere of dry N₂, TEA (0.42 ml, 3 mmol) was added to asuspension of HODhat (0.3282 g, 2 mmol) in 10 ml of dry methylenechloride. The resulting mixture was cooled to 0° C. and a solution ofpivaloyl chloride (0.27 ml, 2.2 mmol) in 5 ml of dry methylene chloridewas introduced dropwise with stirring. The stirring was continued for 30minutes in an ice-bath, and the temperature was allowed to rise to roomtemperature. After 4 hours, the mixture was diluted with 30 ml of CH₂Cl₂and the whole washed with saturated NaHCO₃ (3×20 ml), brine (2×20 ml),and water (2×20 ml), and finally dried over anhydrous MgSO₄. Evaporationof solvent gave a pale yellow solid, which was recrystallized fromEtOAc-hexane to give 0.31 g (6 1%) of the analytically pure esterproduct as colorless needles: mp 137-139° C.; ¹H NMR (CDCl₃): δ 9.16(dd, 1H), 8.57 (dd, 1H), 7.95 (dd, 1H), 1.51 (s, 9H, 3CH₃); IR (KBr):3056 (s), 1797 (vs, COO), 1735 (vs, CONO), 1566 (s), 1479 (m), 1215 (s),1181 (m), 1056 (sh, s), 1016 (s), 974 (m), 831 (s) cm⁻¹.

Anal. Calcd for C₁₁H₁₂N₄O₃: C, 53.21; H, 4.87; N, 22.57.

Found: C, 53.08; B, 4.74; N, 22.51.

EXAMPLE 8

Using the procedure described in Example 8, HODhad, (0.3708 g, 2.3mmol), prepared as described in Example 5, was treated with pivaloylchloride (0.31 ml, 2.53 mmol) in 15 ml of dry CH₂Cl₂ in the presence ofTEA (0.48 ml, 3.45 mmol) to give 0.37 g (65%) of Me₃C—COODhad as a whitesolid. The analytical sample (0.26 g) was obtained after tworecrystallizations from EtOAc-hexane as colorless crystals: mp125-126.5° C.; ¹H NMR(CDCl₃): δ8.91 (dd, 1H), 8.13 (dd, 1H), 8.07 (s,1H, CH), 7.73 (dd, 1H), 1.48 (s, 911, 3CH₃); IR (KBr): 3036 (w), 1794(s, COO), 1715 (vs, CONO), 1583 (sh, m), 1464 (m), 1427 (m), 1296 (s),1059 (vs), 1018 (s), 824 (m) cm⁻¹.

Anal. Calcd for C₁₂H₁₃N₃O₃: C, 58.29; H, 5.29; N, 17.00.

Found: C, 58.34; B, 5.28; N, 17.02.

EXAMPLE 9O-(3,4-Dihydro-4-oxo-5-azabenzo-1,2,3-triazin-3-yl)-1,1,3,3-tetramethyluroniumHexafluorophosphate (HDATU)

Under an atmosphere of dry N₂, 0.22 ml (1 65 mmol) of TEA was added to asuspension of HODhat (0.246 g, 1.5 mmol), prepared as in Example 4, in10 ml of dry CH₂Cl₂. After stirring for 5 min, the resulting lightyellow clear solution was cooled to 0° C. in an ice-bath and 0.4209 g(1.5 mmol) of 1,1,3,3-tetramethyluronium Hexafluorophosphate (TCFH) wasintroduced portion wise with stirring. The stirring was continued for 30minutes in an ice-bath and then at room temperature for 1.5 hours. Theprecipitate which had separated was collected by filtration and washedtwice with methylene chloride and recrystallized twice from MeCN-etherto give 0.42 g (69%) of analytically pure hexafluorophosphate product asa white solid: mp 152° C. (explodes); ¹H NMR(CD₃CN): δ 9.19 (dd, 1H),8.69 (dd, 1H), 8.13 (dd, 1H), 3.21 (s, 12H, 4CH₃); IR (KBr): 1738 (vs),1702 (vs, CON), 1526 (m), 1457 (m), 1412 (s), 1266 (m), 1165 (sh, s),1072 (m), 964 (s), 850 (sh, vs), 708 (s) cm⁻¹.

Anal. Calcd for C₁₁H₁₅N₆O₂PF₆: C, 32.36; H, 3.70; N, 20.58.

Found: C, 32.14; H, 3.79; N, 20.47.

EXAMPLE 10O-(3,4-Dihydro-4-oxo-5-azabenzo-1,3-diazin-3-yl)-1,1,3,3-tetramethyluroniumHexafluorophosphate (HDADU)

Using the procedure of Example 10, the HODhad (0.2447 g, 1.5 mmol),prepared in Example 5, was treated with TCFH (0.4209 g, 1.5 mmol) in 10ml of dry CH₂Cl₂ in the presence of TEA (030 ml, 2.1 mmol) to give 0.55g (90%) of the above-identified hexafluorophosphate product as a whitesolid, which was recrystallized twice from MeCN-ether to give 0.48 g(79%) of analytically pure salt as a white solid: mp 203-205° C. (dec.);¹H NMR (CD₃CN): δ 8.91 (dd, 1H), 8.64 (s, 1H, CH), 8.22 (dd, 1H), 7.88(dd, 1H), 3.17 (s, 12H, 4CH₃); IR (KBr): 1701 (vs, CON), 1605 (sh, m),1528 (s), 1471 (s), 1416 (sh, s), 1266 (s), 1171 (m), 1068 (w), 965 (s),852 (sh, vs), 718 (s) cm⁻¹.

Anal. Calcd for C₁₂H₁₆N₅O₂ PF₆: C, 35.38; 11, 3.96; N, 17.19.

Found: C, 35.51; H, 3.86; N, 17.33.

EXAMPLE 11O-(3,4-Dihydro-4-oxo-5-azabenzo-1,2,3-trazin-3-yl)-1,1,3,3-bis(tetramethylene)-uroniumHexafluorophosphate (HDAPyU)

To a mixture of 0.4923 g (3 mmol) of HODhat as prepared in Example 4 and0.46 ml (3.3 mmol) of TEA in 25 ml of dry CH₂Cl₂ at 0° C., 1.0 g (3mmol) of PyClu was added portion wise with stirring under an atmosphereof dry N₂. Stirring was continued for 1 hour in an ice-bath and then atroom temperature overnight. The clear light yellow mixture was dilutedwith CH₂Cl₂ to 50 ml and washed with ice cold water (2×15 ml) and driedover MgSO₄. The solvent was removed, and the oily residue was dissolvedin 5 ml of MeCN to which 30 ml of ether was added, and the whole wasstored at −20° C. for several days until the oil solidified. The solidwas collected by filtration and redissolved in 20 ml of CH₂Cl₂, and thesolution washed with ice cold water (2×5 ml) and dried over MgSO₄.Removal of solvent gave a pink-yellow solid which was recrystallizedfrom MeCN-ether to give 0.42 g (3 0%) of the above-identified uroniumsalt product as white crystals: mp 136.5° C. (explodes); ¹H NMR (CD₃CN)δ9.17 (dd, 1H), 8.67 (dd, 1H), 8.11 (dd, 1H), 3.75 (t, 8H, 4NCH₂), 1.97(m, 8H, 4CH₂); IR (KBr): 2985 (m), 1734 (vs, CON), 1679 (vs), 1448 (sh,s), 1341 (m), 1169 (m), 1072 (m), 964 (m), 846 (sh, vs) cm⁻¹.

Anal. Calcd for C₁₅H₁₉N₆0₂PF₆: C, 39.13; H, 4.16; N, 18.26.

Found: C, 38.94; B, 4.08; N, 18.30.

EXAMPLE 12[(3,4-Dihydro-4-oxo-5-azabenzo-1,2,3-triazin-3-yl)oxy]tris(pyrrolidino)phosphoniumHexafluorophosphate (PyDAOP)

To a mixture of 0.2462 g (1.5 mmol) of HODhat, prepared as in Example 4and 0.24 ml (1.65 mmol) of TEA in 10 ml of dry CH₂Cl₂ at 0° C., 0.6993 g(1.5 mmol) of PyBrOP (tris(pyrrolidino)bromophosphoniumhexafluorophosphate) was added portion wise with stirring under anatmosphere of dry N₂. Stirring was continued for 1 hour in an ice-bathand then at room temperature overnight. The clear light yellow mixturewas diluted with CH₂Cl₂ to 25 ml and the solution washed with ice coldwater (2×10 ml) and dried over MgSO₄. The resulting light yellow clearsolution was treated with 50 ml of ether and the solid which separatedwas collected by filtration to give 0.45 g (54%) of analytically purePyDAOP as a white solid after recrystallization from MeCN-ether: mp 149°C. (dec.); ¹H NMR (CD₃CN): δ 9.20 (dd, 1H), 8.67 (dd, 1H), 8.13 (dd,1H), 3.42 (td, 12H, 6NCH₂), 1.96 (td, 12H, 6CH₂); IR (KBr): 2982 (m),2893 (m), 1742 (vs, CON), 1566 (m), 1462 (sh, w), 1270 (s), 1224 (s),1140 (s), 1110 (s), 1049 (sh, m), 960 (s), 839 (sh, vs) cm⁻¹.

Anal. Calcd for C₁₈H₂₇N₇0₂P₂F₆: C, 39.34; H, 4.95; N, 17.84.

Found: C, 39.36; H, 5.09; N, 17.90.

EXAMPLE 13

Method A. To a suspension of 0.42 g (3.054 mmol) of HOAt in 20 mL ofanhydrous CH₂Cl₂, there was added 0.43 mL (1 equiv.) of triethylaminewith magnetic stirring. The resulting clear yellow solution was cooledin an ice bath under an atmosphere of N₂ and treated slowly with 0.85 g(1 equiv.) of 2,8-dimethylphenoxaphosphinic chloride.

The reaction mixture was stirred at 0° C. for 30 min, then at roomtemperature for 2 hours. After dilution with 30 mL of CH₂Cl₂, theorganic phase was washed with H₂O, saturated aqueous NaCl (30 mL) anddried over MgSO₄. After removal of solvent with a rotary evaporator withthe aid of a water aspirator, the residue was recrystallized fromCH₂Cl₂-hexane to give 0.65 g (56.3%) of the above-identified phosphinicester as white crystals, mp 164-166° C. (dec); ¹H-NMR (200 MHz, CDCl₃) δ2.472 (s, 6), 7.218-7.559 (m, 5), 8.056-8.130 (d, 2), 8.319-8.368 (d,2), 8.319-8.368 (d, 1), 8.698-8.729 (d, 1). IR (KBr): 1128-(P═O). Anal.Calcd for C₁₉H₁₅N₄PO₃: C, 60.32; H, 4.0; N, 14.81. HREIMS: M+ 378.0882;Found: 378.0877.

Method B: To a suspension of 1.25 g of HOAt in 20 mL of anhydrous CH₂Cl₂there was added 0.623 g (1 equiv.) of imidazole with magnetic stirring.The resulting white suspension was cooled in an ice bath under anatmosphere of N₂ and treated slowly with 2.56 g (1 equiv.) of2,8-dimethylphenoxaphosphinic chloride. The reaction mixture was stirredat 0° C. for 30 min, then at room temperature for 2 hours and dilutedwith 30 mL of CH₂Cl₂. The reaction mixture was filtered in a sinteredglass funnel over anhydrous MgSO₄ under an atmosphere of N₂. Afterremoval of solvent with a rotary evaporator with the aid of a wateraspirator, the residue was recrystallized from CH₂Cl₂-hexane to give2.86 g (82.3%) of the above-identified phosphinic ester as whitecrystals, for which the mp and NMR data agreed with the data reportedabove.

EXAMPLE 14

Using the procedure described hereinabove, the following are alsoprepared:

1.

2. OAt derivatives of

wherein the phenoxophosphinic acid products have the followingsubstituents therein.

(a) 2,8-dibromo (b) 2,8-dichloro (c) 2,8-difluoro (d) 2,8-dimethoxy (e)2,3,7,8-tetramethyl

EXAMPLE 15

Using method B of Example 14, and using 4-Me-HOAt instead of HOAt, theabove-identified compound was made as a white solid.

EXAMPLE 16 Di-o-tolyl-phosphinyloxy-7-azabenzotriazole (DtpOAt)

A. Di-o-toylphosphine oxide. Magnesium turnings (13.96 g) were added to100 ml of anhydrous ether in a 3-neck flask fitted with a condenser,magnetic stirrer and a dropping funnel kept under an atmosphere ofnitrogen. o-Bromotoluene (100 g, 0.579 mol) in 100 ml of ether wasslowly added to the mixture. During the addition, the Grignard reactionwas initiated and became so vigorous that ice bath cooling was neededfrequently. After the addition was complete (40 min), the reactionmixture was refluxed for 15 min and then cooled with an ice bath andtreated slowly with 30.8 ml (0.232 mol) of diethyl phosphite in 40 ml ofether. The mixture was refluxed again for 15 min and cooled with an icebath. Two hundred and fifty milliliters of 10% HCl and 200 ml of waterwere added slowly to the cooled mixture with magnetic stirring. Etherwas evaporated and the insoluble phosphine oxide was collected byfiltration and recrystallized from CH₂Cl₂-hexane (a few drops ofmethanol may be added to help dissolve the solid) to give 39.47 g(73.9%) of the phosphine oxide as a pale yellow solid: mp 94° C., lit.mp 93-94° C., yield 57%; ¹H-NMR (60 MHz, CDCl₃): δ 2.376 (s, 6), 4.234(s, 1), 7.19-7.94 (m, 8); IR (KBr): 2369 (P—H), 1168 (P═O) cm⁻¹.

B. Di-o-toylphosphinic acid. A suspension of 15.04 g ofdi-o-toylphosphine oxide in 80 ml of 5N aqueous NaOH was treated with 40ml of 30% H₂O₂ all at once and the resulting mixture was heated on asteam-bath for 20 min. A clear solution resulted and was filtered whilehot. The filtrate was cooled in an ice bath and acidified slowly withconcentrated HCl, which caused the precipitation of a white solid whichwas recrystallized from MeOH-ether to give 13.4 g (83.3%) of thephosphinic acid: mp 174-176° C., lit. 175-177° C., yield 58-74%; ¹H-NMR(60 MHz, TEA): δ 2.369 (s, 6), 7.245-8.124 (m, 8); IR (KBr): 1143 (P═O)cm⁻¹.

C. Di-o-tolylphosphinic acid chloride (DtpCl). Di-o-tolylphosphinic acid(13.5 g) was slowly added to 50 ml of thionyl chloride with cooling inan ice bath. The mixture was refluxed under a CaCl₂ drying tube for 3hours. After removal of excess thionyl chloride by a water aspirator ina hood, the oily residue was fractionally distilled to give 13.55 g(93.4%) of the phosphinic acid chloride as a colorless oil (bp 158-165°C./0.1 mmHg) which solidified quickly, lit. bp 150-160° C., yield 80.5%;¹H-NMR (60 MHz, CDCl₃): δ 2.449 (s, 6), 7.05-8.08 (m, 8): IR (KBr): 1220(P═O) cm⁻¹.

EXAMPLE 17 Di-o-tolyl-phosphinyloxy-7-azabenzotriazole (DtpOAt)

The above-identified compound was prepared according to the followingscheme which is a variation of the procedure of Example 16:

A. Preparation of Di-o-tolylphosphine oxide (1) Magnesium turnings(13.96 g) were added to 100 mL of anhydrous ether under a nitrogenatmosphere in a 1 L 3-neck flask fitted with an efficient condenser,magnetic stirrer and two dropping funnels. o-Bromotoluene (72 mL) wasadded to the mixture. The rate of addition was adjusted to allow theether to boil slowly. No ice bath cooling was necessary during addition.After the addition was completed the reaction mixture was refluxed for 2hours and then cooled in an ice bath. Diethyl phosphite (30.8 mL) in 50mL of ethyl ether was added from the second dropping funnel and thereaction mixture was stirred at room temperature overnight. Diethylphosphite was distilled before use, bp 62-64° C. (5 mm Hg). Two hundredand fifty milliliters of 10% HCl and 200 mL of water were added slowlyto the cooled mixture and the ether was removed with a rotavap. Theinsoluble material was isolated by filtration, dried and recrystallizedfrom toluene to give 30.7 g (57.4%) of the phosphine oxide as paleyellow crystals, mp 95-97° C., lit. mp 94-95° C.

B. Preparation of di-o-tolylphosphinic acid (2)—Di-o-tolylphosphineoxide (15 g) and 80 mL of 5 N NaOH were treated with 40 mL of 30% H₂O₂.The suspension was heated gently on a steam bath for 20 min. The heatingwas conducted carefully because the reaction could potentially beviolent. Carrying out the reaction in a large beaker avoided loss ofproduct if the reaction became violent. (The reaction mixture doesn'texplode but a lot of foam and gas are developed.) The hot reactionmixture was added to an iced HCl solution (90 mL of conc. HCl plus 30 gof ice). The resulting white solid was isolated by extraction withchloroform (3×75 mL). The chloroform layer was washed with water anddried over magnesium sulfate. After the solvent was removed in vacuumthe resulting solid was recrystallized from 95% ethanol. The aboveamount of phosphine oxide was oxidized in two batches of 15 g each. Theaverage yield of the two runs was 79% for the pure phosphinic acid, mp174-175° C., lit mp 175-177° C.

C. Preparation of di-o-tolylphosphinic acid chloride (3).Di-o-tolylphosphinic acid (22.5 g) was added in one portion to 75 mL ofthionyl chloride and the reaction mixture heated under reflux for 3hours. After removal of excess thionyl chloride, the residual oil wasdissolved in dry dichloromethane (DCM), and the DCM removed at therotavap, the operation being repeated three times. The acid chloridewhich was obtained as an oil which solidified upon standing was usedwithout further purification.

D. Preparation of Dtp-OAt—To a suspension of 12.7 g of HOAt in 300 mL ofanhydrous DCM was added 16.7 mL of DIEA. The resulting clear yellowsolution was cooled in an ice bath under an atmosphere of nitrogen andtreated with 25.8 g of di-o-tolylphosphinic acid chloride dissolved in300 mL of DCM. The reaction mixture was stirred at 0° C. for 1 hour andthen at room temperature for 2 hours. The reaction mixture was dilutedwith 600 mL of DCM and washed with water and saturated aqueous NaCl anddried over MgSO₄. After removal of solvent with a rotary evaporator withthe aid of a water aspirator, the residue was recrystallized fromCH₂Cl₂/ethyl acetate to give 30.1 g (88%) of the active ester as whitecrystals, mp 175-177° C., lit. mp 170-172° C.

It was noted that this material has very low solubility in DMF. Someautomatic peptide synthesizes, for example, the AIB 433 A peptidesynthesizer, requires a solution of the activator in DMF to be placed inone of the reagent bottles which then delivers the activated species tothe reaction vessel. The concentration of the reagent in solution has tobe in the range of 0.3-0.8 M to guarantee the right concentration of theactive species. Since the solubility of this reagent is so low in DMF,the yield for coupling two amino acids to form a peptide was low if thisautomatic synthesizer was used. However, a slight modification of thestructure of the product of this example to make it soluble in DMFenhances its ability to couple amino acids to form peptides. Thefollowing example illustrates a structural modification of Dtp-OAt whichdramatically enhances its solubility in DMF and other organic solvents.

EXAMPLE 18 5-t-Bu-Dtp-Oat

The new coupling reagent was synthesized according to the strategyoutlined hereinbelow. Starting from the commercially available4-t-Bu-toluene the synthesis is completed in 5 steps.

A. Preparation of 2-bromo-4-t-butyltoluene (4). The bromination wascarried-out according to the procedure of Reich, et al. J. Med. Chem.1996, 39, 2781, the contents of which are incorporated by reference. Toa solution of 4-t-butyltoluene (90 mL, 0.518 mol) and two crystals of I₂was added dropwise bromine (27 mL, 0.524 mol). The reaction mixture wasstirred at room temperature for 3 hours and then poured into 1 L of coldwater. The mixture was transferred to a separatory funnel and the lowerlayer was collected. The crude 2-bromo-4-t-butyltoluene was washed withsaturated NaHCO₃, brine and dried over MgSO₄. The crude product wasdistilled under reduced pressure, first using a water aspirator and thenwith a high vacuum oil pump. A first fraction was collected up to b.p.100° C. with the aid of the water aspirator and the residue wasdistilled in high vacuum to give 90.5 g (77%) of the bromide as acolorless liquid, bp 61-65° C. (3-5 mm/Hg).

B. Preparation of bis-(5-t-butyl-2-methylphenyl)phosphine oxide (5).This compound was obtained according to the procedure described forcompound 1, described in Example 17. The phosphine oxide (5) wasrecrystallized from DCM/Hexane to give 15.8 g (32%) of the oxide aswhite crystals, mp 178-180° C.; ¹H-NMR (400 MHz, CDCl₃) δ 1.317 (18H,s), 2.334 (6H, s), 7.715 (2H, dd), 7.480 (2H, dt), 7.779 (2H, dd), 8.215(1H, d, J_(P-H) 474 Hz). IR (KBr) 2336(PH), 1187 and 1168 cm⁻¹ (P═O)Anal. Calcd for C₂₂H₃₁OP: C, 77.16; H, 9.12. Found: C, 77.21; H, 8.98.

C. Preparation of bis-(5-t-butyl-2-methylphenyl)phosphinic acid (6). Toa solution of bis-(5-t-butyl-2-methylphenyl)phosphine oxide (6 g, 17.5mmol) in 60 mL of acetic acid was added slowly 15 mL of 30% H₂O₂. Thereaction mixture was stirred at room temperature for three days and thenpoured into 300 mL of cold water. The white precipitate was isolated byfiltration and recrystallized from ethanol/water to give 6 g (95%) ofpure acid (6) as white crystals, mp 182-183° C.; ¹H-NMR (400 MHz,DMSO-d₆) δ 1.230 (18H, s), 2.172 (6H, s), 7.034-7.902 (6H). IR (KBr)1162 cm⁻¹ (P═O) Anal. Calcd for C₂₂H₃₁O₂P: C, 73.72; H, 8.72. Found: C,74.00; H, 9.01.

D. Preparation of bis-(5-t-butyl-2-methylphenyl)phosphinic chloride (7).The preparation was carried out as described for compound (3) asdescribed in Example 17. The acid chloride was used in the next stepwithout purification. ¹H-NMR (400 MHz, CDCl₃) δ 1.276 (18H, s), 2.384(6H, s), 7.173-7.213 (2H, m), 7.493 (2H dt), 7.903 (2H, dd), IR (KBr)1231 cm⁻¹ (P═O).

E. Preparation ofbis-(5-t-butyl-2-methylphenyl)phosphinyloxy-7-azabenzotriazole. To asuspension of 0.76 g (5.58 mmol) of HOAt in 30 mL of anhydrous DCM wasadded 0.78 mL (5.6 mmol) of TEA. The resulting clear yellow solution wascooled in an ice bath under an atmosphere of nitrogen and treated with 2g (5.27 mmol) of bis-(5-t-butyl-2-methylphenyl)-phosphinic acid chloridedissolved in 20 mL of DCM. The reaction mixture was stirred at 0° C. for1 hour and then at room temperature for 45 hours. The reaction mixturewas diluted with 40 mL of DCM and washed with water and saturatedaqueous NaCl and dried over MgSO₄. The solvent was removed with a rotaryevaporator with the aid of a water aspirator. The above product wasobtained as a white solid [2.3 g (86%)] after recrystalization fromhexane and then from hexane containing a small amount of ethyl acetate:mp 86-87° C., ¹H-NMR (400 MHz, CDCL₃) δ 1.271 (18H, s), 2.687 (6H, s),7.264-7.298 (2H, m), 7.369 (1H, dd), 7.545 (2H dt), 7.974 (2H, dd),8.309 (1H, dd), 8.729 (1H, dd). IR (KBr) 1242 cm⁻¹ (P═O) HRFAB MS forC₂₇H₃₄N₄O₂P, M⁺ 477.2419, found 477.2400.

Small-scale solubility tests showed that this compound (t-Bu-Dtp-OAt)was highly soluble in DMF and thus could be used in the automatedpeptide synthesizer.

As indicated hereinabove, without wishing to be bound, it is believedthat during peptide coupling using uronium or phosphonium salts, theN-protected carboxylic acid first reacts with the coupling reagent toform an active ester, which then reacts with the amino component to givethe corresponding amide. Therefore, the speed of formation of such anactive ester is one of the important factors in evaluating theefficiency of the coupling reagent. The model chosen hereinbelowinvolved conversion of N-benzyloxycarbonyl-α-aminoisobutyric acid(CBZ-Aib-OH) to the corresponding active ester in both DMF and CDCl₃, asdescribed in the chemical equation hereinbelow.

In the study, a comparison was made with derivatives of HOBt, HOAt withHODhbt:

The benzylic CH₂ units of CBZ-Aib-OH (55.09) and active ester (65.20)were monitored by NMR. Assignment of the peak at 65.20 in the activeesters was confirmed by authentic synthesis.

Because of the sterically hindered carboxyl group of Aib, activation inthe above equation is slow relative to the case of the proteinogenicamino acids allowing different coupling reagents to be more closelydifferentiated.

The procedure is described in Example 19 hereinbelow.

EXAMPLE 19

(a) Di-o-tolylphosphinyloxybenzotriazole (DtpOBt). The preparation wascarried out similarly to that as described for preparing the product ofExample 16: HOBt (0.125 1 g, 1 mmol) was treated with Dtp-Cl (0.267 g, 1mmol) in the presence of DIEA (0.21 ml, 1.2 mmol) for 5 hours in 10 mlof dry CH₂Cl₂ to give 0.24 g (66%) of white solid after work-up.Recrystallization from CH₂Cl₂-hexane gave 0.18 g of an analytically puresample of the ester as colorless crystals: mp 198-200° C.; ¹H-NMR(CDCl₃): δ8.06 (m, 3), 7.30 (m, 6), 2.62 (s, 6); IR (KBr): 3065 (W),1593 (s), 1457 (s), 1362 (m), 1278 (m), 1230 (vs), 1151 (s), 1084 (sh,s), 812 (vs), 774 (sh, vs), 704 (m) cm⁻¹. Anal. Calcd for C₂₀H₁₈N₃O₂P:C, 66.10; H, 4.92; N, 11.56. Found C, 65.75; H, 4.97; N, 11.41.

(b) 3-[di-o-tolyl)phosphinyloxy]-3,4-dihydro-4-oxo-2,3 benzotriazine](DtpODhbt) was made according to method A described for DmppOAt (MethodA) in Example 14 using HODhbt, which was prepared similarly to themethod described in Ex. 4 using HOBt instead of HOAt and usingdi-o-tolylphosphinic chloride. The product was obtained as whitecrystals (76.5%) after recrystallization from ethyl acetate-ether: mp178-179° C. (dec); ¹H-NMR (200 MHz, CDCl₃): δ2.58 (s, 6), 7.28 (m, 4),7.487-7.570 (m, 2), 7.75 (m, 1), 7.90-8.15 (m, 4), 8.351 (dd, 1); IR(KBr): 1709 (C═O), 1240 (P═O) cm⁻¹. Anal. Calcd for C₂₁H₁₈N₃PO₃; C,64.45; H, 4.64; N, 10.74. Found: C, 64.49; H, 4.54; N, 10.69.

(c) Active Ester Formation. To a solution of 0.1 mmol of CBZ-Aib-OH and0.1 mmol of the appropriate coupling reagent in 0.5 ml of CDCl₃ or DMF,was added 0.1 mmol of DIEA. The mixture was immediately transferred toan NMR tube which was placed in the probe of a Hitachi R-1200 (60 MHz)NMR instrument. Integration of the ¹H-NMR peaks at δ5.1 (acid) and 5.2(active ester) as the reaction progressed at the probe temperature (37°C.) allowed for rough determination of the relative rates. The resultsare given in Table 1 and represent the averages of at least two runs.

t_(1/2) (DMF), t_(1/2) (CDCl₃), Coupling Reagent min min DEPOAt <2 2–3DPOPOAt <2 2–3 DEPDBt 7–8 45–47 DPOPDBt <2 <2 DtpOBt 65–70 11–12 hN-HATU <2 14–15 N-HAPyU <2 <2 HDTU <2 <2 N-HBTU <2 >24 h

In the examples that follow, it is to be understood that the amino acidsequence is presented in the amino to carboxy direction, from left toright.

EXAMPLE 20

To test for configuration control, three different peptides,CBZ-Phe-Val-Pro-NH₂, CBZ-Gly-Phe-Pro-NH₂, andCBZ-Gly-Gly-Val-Ala-Gly-Gly-OMe (SEQ ID NO:1) were prepared and the lossof configuration during the coupling was determined. An exemplaryprocedure is given for a dipeptide as follows:

A. CBZ-Phg-Pro-NH₂ As a standard protocol, 35.6 mg (0.125 mmol) ofCBZ-Phg-OH, 14.3 mg (0.125 mmol) of H-Pro-NH₂, and 0.25 mmol of base in1 ml of DMF or other solvent was treated with 0.125 mmol of anappropriate coupling reagent at 0° C. The mixture was stirred at 0° C.for 1 hour and at room temperature overnight. The resulting mixture wasdiluted with 25 ml of EtOAc and washed with HCl (2×10 ml), 10% NaHCO₃solution (2×10 ml) and brine (2×10 ml), and dried over MgSO₄. An oilypeptide residue was obtained after removing solvent. The oily residuewas redissolved in 1 ml of CH₂Cl₂ and 20 ml of hexane was added. A whitesolid was obtained after filtration. About 5 mg of this crude product,usually containing both LL- and DL-forms of CBZ-Phg-Pro-NH₂, wasdissolved in 4 ml of MeCN and directly analyzed by HPLC as described inWenschuh, et al., J. Org. Chem., 1995, 62, 405, the contents of whichare incorporated by reference.

B. CBZ-Phe-Val-Pro-NH₂. The standard protocol as described above forCBZ-Phg-Pro-NH₂ was followed.

C. CBZ-Gly-Phe-Pro-NH₂. The standard protocol as described above forCBZ-Phg-Pro-NH₂ was followed.

D. CBZ-Gly-Gly-Val-Ala-Gly-Gly-OMe (SEQ ID NO 1.) As described forCBZ-Phg-Pro-NH₂, a solution of 45.6 mg (0.125 mmol) ofCBZ-Gly-Gly-Val-OH, 31.7 mg (0.125 mmol) of H-Ala-Gly-Gly-OMe.HCI and49.6 μl (0.375 mmol) of TMP in 1 ml of DMF was treated with 0.125 mmolof an appropriate coupling reagent at 0° C. The resulting mixture wasstirred at 0° C. for 1 hour and then at room temperature overnight. Thecrude hexapeptide methyl ester was isolated by evaporation of solventfollowed by direct column chromatography using MeOH/CHCl₃/HOAc (3:7:0.1)as eluate. The crude material from the column, containing both LL- andDL-forms of hexapeptide, was examined by HPLC as described in Carpino,J. Org. Chem., 1994, 59, 695, the contents of which are incorporated byreference.

The results are given in Table 2

TABLE 2 Effect of Coupling Reagent on Loss of Configuration During [2 +1] Coupling Leading to CBZ-Phe-Val-Pro-NH₂, CBZ-Gly-Phe-Pro-NH₂ and [3 +3] Coupling Leading to CBZ-Gly—Gly-Val-Ala- Gly—Gly-OMe in DMF with 2eqs. TMP as base. Coupling CBZ-Phe-Val- CBZ-Gly-Phe-CBZ-Gly—Gly-Val-Ala- Reagent Pro-NH₂ Pro-NH₂ Gly—Gly-OMe DEPOAt 0.9(0.9) <0.1 <0.1 DmppOAt 3.6 (2.0) 0.3 DtpOAt 2.9 (1.4) 0.4 N-HATU 5.0(1.8) 1.1 (0.9) 2.4 DEPDBt  3.5 0.3 2.4 DtpODhbt 4.3 (3.6) HDTU 8.5(4.0) 3.3 DtpOBt 11.4 2.2 N-HBTU 14.2 3.6 8.2 ^(a)Figures in parenthesisrefer to identical runs but with 1 eq. of the appropriate HOXt e.g.,HOAt, HOBt or HODhbt added.

As confirmed by the data herein, the new phosphorous-based OAtderivatives of the present invention are much more effective inpreserving configuration than any of the other tested reagents,including N-HATU. The best of the previously-describeduronium/guanidinium salts (N-HAPyU) sometimes equals the results of thenew phosphorus esters, but where differences are observed, the latterhave proved more effective in every case examined to date.

Among the results obtained from these data involving N-HAPyU, it wasfound that for the new reagents DEPOAt and DPOPOAt, a one-equivalentexcess of proline serving as base gave the lowest epimerization levelsyet observed for the tripeptide CBZ-Phe-Val-Pro-NH₂ in DMF (0.5%LDL-isomer). Upon switching to other solvents, even greater differenceswere found between the new phosphorus reagents of the present inventionand the related uronium/guanidinium salts. For example, in the specialstructure-breaking combination solvent trifluoroethanol/trichloromethane(TFE, TCM, 1:3), 12.2% of the LDL-form was obtained for DPOPOAt/TMP asopposed to 38.5% for N-HATU/TMP. In CH₂Cl₂ in the presence of TMP, 2.2%(DEPOAt) and 2.9% (DPOPOAt) were clearly better than values observed forguanidinium reagents N-HATU (9.3%) and N-HAPyU (5.3%).

In order to determine the coupling efficiency of diphenylphosphorochloridate (DPOPCl) various coupling conditions were used. Itwas noted that without additive, DPOPCl gave only a very small amount ofthe desired peptide for both diisopropylethylamine andtrimethylpyridine. If one equivalent of HOAt(1-hydroxy-7-azabenzotriazole) was present, the results were acceptable.Indeed the mixture DPOPCl/HOAt/Base, which contains DPOPOAt as theactive species, gave results which are comparable to those obtained withthe isolated reagent DPOPOAt.

The Tripeptide CBZ-Phe-Val-Pro-NH₂ was also chosen as a model to studyloss of configuration associated with use of various reagents of thepresent invention under solid phase conditions. In comparison withresults obtained in solution, the data show how much more difficult itis to maintain configuration in the solid phase mode. The systeminvolved overnight coupling of four equivalents of CBZ-Phe-Val-OH ontoH-Pro-PAL-PEG-PS in the presence of 8 eqs. of trimethylpyridine in DMF,cleavage of the tripeptide from the resin via trifluoroacetic acid/H₂0(9:1) over a period of 1 hour and separation of the diastereomers asdescribed for the solution system. Although extensive loss ofconfiguration occurs in all cases, the data show that the effectivenessof the various coupling reagents follows the same order as in solution,thus coupling reagent/LDL (%): DEPOAt/11.6, N-HAPyU/13.0, N-HATU/13.6,DPOPODBt/19.4, DEPDBt/19.5, HDTU/24.2, N-HBTU/29.8.

EXAMPLE 21 ACP Assembly Via Stepwise Coupling on Solid Phase

In order to demonstrate the suitability of the organophosphorus-basedcoupling reagents and compare their performance with that of thecorresponding uronium/guanidinium analogs in solid phase syntheses,several syntheses of the ACP decapeptide segment 65-74(H-Val-Gln-Ala-Ala-Asp-Tyr-Ile-Asn-Gly-NH₂) (SEQ ID NO:2) were carriedout.

The protocol is as follows: 150 mg of Fmoc-Gly-PAL-PEG-PS resin (0.19mmol/g, 0.0285 mmol) in a 10-ml disposable syringe fitted with a Teflonfilter was washed with CH₂Cl₂ (3×10 ml) and DMF (3×10 ml) anddeprotected with 20% piperidine in DMF (10 ml) for 7 min. Thedeprotected resin was washed with DMF (3×10 ml), CH₂Cl₂ (3×10 ml) andagain DMF (3×10 ml). Preactivation was carried out for 7 min using 25.5mg (0.04 mmol, 1.5 equiv) of Fmoc-Asn(Trt)-OH, 15.75 mg (0.04 mmol, 1.5equiv) of DPOPOAt and 14.89 μl (0.09 mmol, 3 equiv) of DIEA(diisopropylethylamine) in 0.15 ml of DMF in a 4-ml vial. Following therequisite preactivation period (7 min), the solution of the activatedamino acid was added to the resin. The small vial was washed with 0.04ml of DMF, and the washing was also added to the above resin. Theresulting resin mixture was allowed to react at room temperature for 1.5min. The loaded resin was washed with DMF (3×10 ml) and the Fmoc groupwas deblocked with 10 ml of 20% of piperidine in DMF for 7 min. Washingthe deblocked resin with DMF (3×10 ml), CH₂Cl₂ (3×10 ml) and DMF (3×10ml) was followed by an analogous coupling step with Fmoc-Ile-OH. Otheramino acids were coupled similarly and after the last coupling withFmoc-Val-OH and deblocking of the Fmoc group with 20% piperidine in DMF,the loaded resin was washed with DMF (3×10 ml), CH₂Cl₂ (3×10 ml), EtOH(5 ml) and ether (5 ml). The resin was then treated with 10 ml of 90%aqueous trifluoroacetic acid for 2 hours, filtered, and washed on thefilter with 10 ml of 10% trifluoroacetic acid in CH₂Cl₂ and 10 ml ofCH₂Cl₂. The combined filtrates were evaporated to dryness. The crudeproduct was washed four times with anhydrous ether and separated bycentrifugation. The yield was calculated by the weight of the crudeproduct. For analysis 1 mg of the crude product was dissolved in 1 ml of0.1% aqueous trifluoroacetic acid and injected directly onto the HPLCcolumn for analysis. The procedure was repeated using the same couplingagent until the peptide of SEQ ID NO. 2 SEQ ID NO:2 was prepared.

This procedure was repeated using each of the coupling agents listed inTable 3 for the preparation of the peptide of SEQ. ID. NO. 2 SEQ IDNO:2. The results are given in Table 3.

TABLE 3 Distribution of Products, Including Various Deletion Peptides,According to HPLC Analysis for the Assembly of ACP (65–74) via HOAt- andHODhbt-Based Coupling Reagents Coupling Equiv. of Preactivation CouplingACP -2Ile -Ile72 -Ile69 -Val -Ala -Asn Entry Method Reagents Time (mm)(min) (%) (%) (%) (%) (%) (%) (%) 1 DEPOAt 1.5 7 1.5 84 — 2 2 1 4 5 2DPOPOAt 1.5 7 1.5 85 — 2 1 2 6 3 3 DEPDBt 1.5 7 1.5  6 9 13 19 3 — 1 4DPOPOBt 1.5 7 1.5 23 21 26 19 1 1 2 5 N-HATU 1.5 7 1.5 85 — 1 1 3 — 10 6 HDTU 1.5 7 1.5 38 15 15 26 — — 5 7 DEPOAt 1.5 0 1.5 86 — 4 2 2 3 1 8DPOPOAt 1.5 0 1.5 81 — 4 1 1 7 — 9 DEPDBt 1.5 0 1.5  <1^(c) — — — — — —10 DPOPOBt 1.5 0 1.5 29 17 25 17 2 — 3 11 N-HATU 1.5 0 1.5 87 — 3 1 2 —6 12 HDTU 1.5 0 1.5 30 15 19 22 3 — 4 *A reversed-phase C-18 column wasused with elution by a linear gradient over 20 mm of 0.1% TFA in MeCNand 0.1% TFA from 1:19 to 1:1, flow rate 1.0 mI/mm. ^(b) Couplings werecarried out in DMF in the presence of 2 equivalents ofdiisopropylethylamine per equivalent of Fmoc-amino acid/couplingreagent. ^(c)Only a trace of the desired product was obtained.

In this experiment, coupling times are shortened and excesses ofreagents are reduced in order to bring out differences among the variousreagents studied. Under these conditions, incomplete incorporations weredetected for Asn onto Gly, Ile onto Asn, Ile onto Asp, Val onto Gln andAla onto Ala or Asp. Analysis of the chromatograms indicated that thenew HOAt-based organophosphorus reagents are as effective as N-HATUunder these so-called “1.5×1.5” conditions with or withoutpreactivation. Under normal coupling conditions such as 4 eqs excessamino acid/30 min. coupling time, all reagents worked well with theexception of HDTU.

EXAMPLE 22

In the following example, reactions of the hindered active estersCBZ-Aib-OXt with p-chloroaniline (PCA) were studied in CDCl₃.Approximate halftimes were determined by proton NMR analysis accordingto the disappearance of the benzylic CH₂ unit (δ 5.2) of the activeesters and appearance of the benzylic CH₂ residue (δ 5.5) of theproduct.

CBZ-Aib-OXt Esters. The reaction of Z-Aib-ODhat with PCA is taken as anexample to demonstrate the standard method used in order to followaminolysis via an NMR protocol: To a solution of 47.9 mg (0.125 mmol) ofCBZ-Aib-ODhat in 0.5 ml of CDCl₃, was added 15.6 mg (0.125 mmol) ofp-chloroaniline (PCA). The mixture was immediately transferred to an NMRtube, which was placed in the probe of a Hitachi R-1200 (60 MH_(z))instrument. Integration of the ¹H NMR peaks at δ 1.8 (CH₃ residue ofester CBZ-Aib-ODhat) and 1.57 (CH₃ residue of amide CBZ-Aib-PCA) [orpeaks at δ5.2 (benzylic CH₂ unit of ester CBZ-Aib-ODhat) and 5.05(benzylic CH₂ unit of the product amide)] as the reaction progressed atthe NMR probe temperature (37° C.) allowed for rough determination ofthe relative rates. The results given in Table 4 are the average of atleast two runs.

TABLE 4 Approximate Halftimes for Disappearance of CBZ-Aib-OXt in CDCl₃in the Presence of p-Chloroaniline CBZ-Aib-OXt t_(1/2) (min)CBZ-Aib-ODhat 8–9 CBZ-Aib-OAt  9–10 CBZ-Aib-ODhbt 12–13 CBZ-Aib-ODhad 70 CBZ-Aib-OBt 210

It was found that the ODhat ester is slightly more reactive even thanthe OAt ester, which was previously found to be the most reactivederivative among these esters. Interestingly, despite the structuralsimilarity between HODhat, i.e.,3-hydroxy-4-oxo-3,4-dihydro-5-azabenzo-1,2,3-triazine, and HODhad, i.e.,3-hydroxy-4-oxo-3,4-dihydroxy 5-azabenzo-1,3-diazine the reactivities ofthe corresponding active esters are very different. Without wishing tobe bound, it is believed this may be due to the presence or absence ofadditional neighboring group effects promoted by the presence or absenceof a nitrogen atom substituted at the 2-position. On the other hand,comparison of the OBt and ODhad esters demonstrates the importance ofthe neighboring carbonyl group.

EXAMPLE 23

In this example, a second model was used. Pivaloyl esters in thepresence of various comparative coupling agents were treated withbenzylamine and N-methylbenzylamine, which led to the formation ofamides of the following formula:

Approximate halftimes for these reactions were determined by proton NMRanalysis, according to the disappearance of the methyl peak (δ 1.5) forpivaloyl-OXt and the appearance of the methyl peak for products XXV_(a)(δ 1.2) or XXV_(b) (δ 1.3). The protocol is as follows:

Me₃CCOOXt Esters. As in the case with Example 22, the reaction ofpivaloyl ester with N-methyl benzylamine is taken here as an example todemonstrate the methodology used: To a solution of 31.0 mg (0.125 mmol)of Me₃CCOODhat in 0.5 ml of CDCl₃, was added 15.1 mg (16.1 μl, 0.125mmol) of PhCH₂NHMe. The mixture was immediately transferred to an NMRtube, which was placed in the probe of a Hitachi R-1200 (60 MHz)instrument. Integration of the ¹H NMR peaks at δ 1.5 (CH₃ residue ofester Me₃CCOODhat) and 1.3 (CH₃ residue of amide) as the reactionprogressed at the NMR probe temperature (˜37° C.) allowed for roughdetermination of the relative rates. The results given in Table 5 arethe average of at least two runs.

The results are tabulated in Table 5.

TABLE 5 Approximate Halftimes for Disappearance of Me₃C—CO-Oxt in CDCl₃Me₃CCOOXt t_(1/2) (PhCH₂NH₂) t_(1/2) (PhCH₂NHMe)) Me₃CCOODhat <1 min <2min Me₃CCOOAt <1 min 7–8 min Me₃CCOODhbt <1 min 18–20 min Me₃CCOODhad <1min 35–40 min Me₃CCOOBt <1 min 4.5 hr

In the case of benzylamine all reactions were rapid whereas in the caseof the more hindered N-methyl derivative, clear reactivity differenceswere seen according to the following order: ODhat>OAt>ODhbt>ODhad>OBt.Again the greater reactivity of the HODhat ester relative to thatderived from HOAt is seen.

EXAMPLE 24

Another comparative study was run to compare relative rates of couplingprocesses involving the reaction of CBZ-Aib-OH with p-chloroaniline(PCA) in the presence of a coupling reagent. Because formation ofintermediate Z-Aib-OXt is usually very fast, halftimes are determined bydisappearance of the benzylic CH₂ residue (δ 5.2) of the active esterand appearance of the benzylic CH₂ unit (δ 5.05) of product CBZ-Aib-PCAunless otherwise noted. The protocol for the preparation of this productis the same as described in Example 23. Approximate halftimes arecollected in Table 6. In this case various solvent systems wereexamined.

TABLE 6 Approximate Halftimes for Disappearance of [CBZ-Aib-Oxt] inVarious Solvent Systems in the Presence of p-Chloroaniline Couplingt_(1/2) t_(1/2) t_(1/2) t_(1/2) Reagent (CDCl₃) (CD₃CN) (DMF)(DMF/CDCI₃)^(a) HDATU <3 min 70–75 min 30–40 min N-HATU 18–24 min 90–95min 75–85 mm 40–45 min HDTU 20–25 min 4.5–5 hr 6–7 hr 2.5–3 hr N-HBTU3.5–4 hr 10–11 hr^(b) ^(a)1:1 mixture of DMF/CDCl₃. ^(b)In this case,the halftime is determined by disappearance of acid Z-Aib-OH andappearance of both intermediate active ester and amide CBZ-Aib-PCA.

Interestingly in all solvent systems examined except for DMF, the newcoupling reagent was found to be more reactive than N-HATU. In CDCl₃,HDATU is at least six times as reactive as N-HATU and about eight timesas reactive as HDTU. So far, in every case tested HDATU was shown to besignificantly more reactive than HDTU.

Example 25

In order to test the configuration-retention effectiveness of theadditives HODhat and HODhad, and the coupling reagents HDATU, HDADU,HDAPyU, HDPyU, PyDAOP, and PyDOP, the following model peptide systemswere examined. These involve a [1+1] stepwise coupling, and three [2+1]and one [3+3] segment couplings.

CBZ-Phg-Pro-NH₂, XXVI CBZ-Phe-Val-Pro-NH₂, XXVII CBZ-Gly-Phe-Pro-NH₂XXVIII CBZ-Gly-Gly-Val-Ala-Gly-Gly-OMe, (SEQ ID NO 1) XXIX andH-Gly-Leu-Phe-OBzl XXX

Test couplings were carried out as described previously in Example 22and in L. A. Carpino, et al., J. Org. Chem. 1990, 61, 2463, forCBZ-Phg-ProNH₂, CBZ-Phe-Val-Pro-NH₂, CBZ-Gly-Phe-Pro-NH₂ andCBZ-Gly-Gly-Val-Ala-Gly-Gly-OMe substituting the coupling reagent listedhereinbelow in the tables and the protocols therein, the results ofwhich are incorporated by reference. For Boc-Gly-Leu-Phe-OBzl,(Bzl=benzyl) 60.6 mg (0.21 mmol) of Boc-Gly-Leu-OH, 85.45 mg (0.20 mmol)of H-Phe-OBzl.pTsOH and 0.22 mmol of an appropriate coupling additive(HOXt) were dissolved in 1 ml of DMF or trifluroethanol/trichloromethane(1:3 v/v). To the mixture, a solution of 34.2 mg (0.22 mmol) of EDC(1-ethyl-3-3′-(dimethylamino)-propyl)carbodiimide in 1 ml of DMF ortrifluroethanol/trichloromethane was added and the whole mixture wasstirred at room temperature overnight. The resulting mixture was dilutedwith 25 ml of EtOAc and washed with 1 N HCl (2×10 ml), 10% NaHCO₃ (2×10ml) and brine (2×10 ml), and dried over MgSO₄. After removal of solvent,the solid was weighed to determine the yield. The solid was then stirredwith 2 ml of 50% trifluoroacetic acid in a methylene chloride solutionfor 2 hours to deblock the BOC-group. The trifluoroacetic acid andCH₂Cl₂ were then removed in vacuo and 20 ml of anhydrous ether was addedto the oily residue, and the mixture was stored at room temperatureovernight. The white precipitate which had separated was collected byfiltration and washed with ether. About 5 mg of the crude product,containing both LL- and DL-forms of XXX was dissolved in 4 ml of MeCNand directly analyzed by HPLC using a reversed-phase Waters C-18 column,with elution by a linear gradient over 20 min of 0.1% trifluoroaceticacid in MeCN and 0.1% aqueous TFA from 1:9 to 11:9, at a flow rate of1.0 ml/min. The retention times for the LL and DL-forms of XXX were 17.3and 17.9 min, respectively. The results are as follows:

For the sensitive coupling of the urethane-protected CBZ-Phg-OH toH-Pro-NH₂ to give XXVI, HDATU was more effective in preservingconfiguration than HDTU and N-HBTU, but not better than N-HATU.Curiously with this system, use of the base diisopropylethyl amine(DIEA) proved more satisfactory than collidine (TMP), a result that israrely observed in the case of the corresponding segment couplings.Results are collected in Table 7.

TABLE 7 Effect of Coupling Regeant, Base and Solvent on the Preservationof Configuration during the Formation of XXVI via [1 + 1] CouplingCoupling Base Reagent (%) Additive (Eq.) Solvent Yield (%) DL HDATUDIEA(2) DMF 83.9 4.8 HDTU DIEA(2) DMF 78.4 12.8 N-HATU DIEA(2) DMF 71.72.8 N-HBTU DIEA(2) DMF 81.3 6.3 HDATU TMP(2) DMF 87.5 6.0 HDTU TMP(2)DMF 80.7 16.0 N-HATU TMP(2) DMF 90.8 3.8 N-HBTU TMP(2) DMF 85.4 8.7 DCCHODhat(1) TMP(1) TFE/TCM^(a) 74.8 0.4 DCC HODhbt(1) TMP(1) TFE/TCM^(a)71.8 0.8 DCC HOAt(1) TMP(1) TFE/TCM^(a) 69.2 0.3 ^(a)In this case, 1.3ml of trifluoroethanol-chloroform (1:3 v/v) was used as solvent.

With carbodiimide reagents in the solventtrifluroethanol/trichloromethane, HODhat was even more effective thanHODhbt. Thus, EDC/HODhat gave 0.5% of the DL-isomer, whereas EDC/HODhbtled to 1.3% of the same form. For DCC/HODhat and DCC/HODhbt in thepresence of 1 equivalent of trimethylpyridine, the figures were 0.4% and0.8%, respectively.

For the well-studied segment coupling of CBZ-Phe-Val-OH to H-Pro-NH₂leading to tripeptide XXVII, the results are tabulated in Table 8.

TABLE 8 Effect of Coupling Reagent, Base and Solvent on the Preservationof Configuration during the Formation of XXVII via [2 + 1] CouplingCoupling Base Yield LDL Reagent Additive (Eq.) Solvent (%) (%) HDATUDIEA(2) DMF 85.4 15.1 HDTU DIEA(2) DMF 81.0 13.3 HDADU DIEA(2) DMF 72.427.6 N-HATU DIEA(2) DMF N-HBTU DIEA(2) DMF HDATU TMP(2) DMF 88.8 8.7HDTU TMP(2) DMF 86.4 8.5 HDADU TMP(2) DMF 83.8 18.6 N-HATU TMP(2) DMF80.1 N-HBTU TMP(2) DMF 81.2 HDATU HODhat(1) TMP(2) DMF 68.0 7.3 HDTUHODhbt(1) TMP(2) DMF 65.0 4.0 N-HATU HOAt(1) TMP(2) DMF * DIEA =diisopropylethylamine; TMP = trimethylpyridine

For the rather insensitive case of the segment coupling of Z-Gly-Phe-OHto H-Pro-NH₂, the results paralleled those for XXVII. Results arepresented in Table 9.

TABLE 9 Effect of Coupling Reagent, Base and Solvent on the Preservationof Configuration during the Formation of XXVIII via [2 + 1] Coupling^(a)Coupling Base Solvent Yield LDL Reagent Additive (Eq.) Solvent (%) (%)HDATU DIEA(2) DMF 96.5 0.5 HDTU DIEA(2) DMF 82.0 1.5 HDADU DIEA(2) DMF92.8 7.2 N-HATU DIEA(2) DMF 86.0 0.8 N-HBTU DIEA(2) DMF 84.8 5.9 PyDAOPDIEA(2) DMF 89.9 0.8 PyDOP DIEA(2) DMF 94.3 0.7 HDATU TMP(2) DMF 94.11.0 HDTU TMP(2) DMF 98.3 1.7 HDADU TMP(2) DMF 86.6 8.4 N-HATU TMP(2) DMF96.5 1.1 N-HBTU TMP(2) DMF 88.2 3.6 HDAPyU TMP(2) DMF 78.8 1.2 HDPyUTMP(2) DMF 80.9 1.4 PyDAOP TMP(2) DMF 84.3 1.8 PyDOP TMP(2) DMF 90.4 1.7HDATU DIEA/TMP^(b) DMF 85.8 0.8 HDTU DIEA/TMP^(b) DMF 84.8 1.5 N-HATUDIEA/TMP^(b) DMF 76.4 0.9 DIC(diisopropylcarbodimide) HODhat(1) DMF 86.20.5 DIC HODhbt(1) DMF 81.4 0.8 DIC HODhad(1) DMF 74.3 8.2 DIC HOAt(1)DMF 82.3 0.4 EDC HCl HODhat(1) TMP(1) DMF 94.0 1.8 EDC HCl HODhad(1)TMP(1) DMF 85.2 2.2 EDC HCl HOAt(1) TMP(1) DMF 89.3 3.3 EDC HClHODhat(1) TMP(2) DMF 90.1 2.8 EDC HCl HODhbt(1) TMP(2) DMF 86.1 1.6 EDCHCl HOAt(1) TMP(2) DMF 91.0 1.7 EDC HODhat(1) DMF 88.8 3.4 EDC HODhat(1)TFE/TCM^(c) 80.2 2.5 ^(a)Abbreviations as given hereinabove. ^(b)In thiscase, a combination of 1 equiv of DIEA and 1 equiv of TMP was used asbase. ^(c)1.3 ml of trifluoroethanol-chloroform was used as solvent.

For the preparation of tripeptide XXVIII, HDATU was similar to or evenslightly more effective than N-HATU.

With respect to the test tripeptide XXX, the coupling of H-Phe-OBzlTosOH with Boc-Gly-Leu-OH in the presence of EDC/additive (couplingagent) in various solvents gave a product Boc-Gly-Leu-Phe-OBzl which wasBOC-deblocked via 50% TFA/CH₂Cl₂ to give the crude tripeptide, which wasdirectly analyzed by HPLC.

In the EDC-mediated synthesis of XXX carried out intrifluoroethanol/chloroform (1:3 v/v), the three additives HODhat,HODhbt, HOAt were found equally effective with less than 0.1%epimerization being observed. Upon switching to DMF as solvent,differences, although small, could be noted. Results are shown in Table10.

TABLE 10 Effect of Coupling Reagent, Base and Solvent on thePreservation of Configuration during the Formation of XXX via [2 + 1]Coupling Coupling Reagent Solvent Yield LDL EDC/HODhat DMF 76.2 0.20EDC/HODhbt DMF 88.5 0.25 EDC/HOAt DMF 90.6 0.33 EDC/HOBt DMF 77.4 0.43EDC/HODhat TFE/TCM^(b) 98.6 <0.1 EDC/HODhbt TFE/TCM^(b) 96.2 <0.1EDC/HOAt TFE/TCM^(b) 98.2 <0.1 EDC/HOBt TFE/TCM^(b) 90.0 0.20^(b)Combination solvent trifluoroethanol-chloroform (1:3 v/v) was used.

Following preliminary studies with simple di- and tripeptide models,XXVI, XXVII, XXVIII and XXX, a test peptide XXI was assembled. Thecoupling of Z-Gly-Gly-Val-OH to H-Ala-Gly-Gly-OMe is a sensitive testfor the nature of both coupling reagent and base. Results for thereaction in DMF, in the presence of collidine are gathered in Table 11.HDATU was found to be more effective in preventing loss of configurationat valine than N-HATU and other coupling reagents. Epimerization levelsup to 8.2% of the DL-form were noted according to the order:HDATU<N-HATU<HDTU<N-HBTU.

TABLE 11 Effect of Coupling Reagent, Base and Solvent on thePreservation of Configuration during the Formation of XXIX via [3 + 3]Coupling Coupling Reagent Base Solvent Yield (%) LDL (%) HDATU TMP(3)DMF 98.4 0.8 HDTU TMP(3) DMF 95.0 3.3 N-HATU TMP(3) DMF 96.6 2.4 N-HBTUTMP(3) DMF 85.6 8.2

EXAMPLE 26

In order to demonstrate the suitability of the new HODhat-based couplingreagent HDATU and compare its performance with that of the correspondingguanidinium/uronium analogs N-HATU and HDTU in solid-phase syntheses, 30syntheses of the ACP segmentH-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-GlyNH₂, (SEQ ID NO:2) were carriedout by an Fmoc/tert-butyl protection scheme as described in Example 22.Polyethylene glycol-polystyrene (PEGPS)-resin bearing Fmoc-glycine wasused as solid support. Peptide elongation was performed manually,coupling times being shortened and excesses of reagents being reduced inorder to bring out the differences among the various coupling reagentsstudied. Under these conditions, incomplete incorporations were detectedfor Asn onto Gly, Ile onto Asn, Ile onto Asp, and Val onto GIn. Peptidepurity was judged by reverse-phase HPLC analysis, after cleavage fromthe resin with TFA-H₂O (9:1) for 2 hours at room temperature. Theresults are collected in Table 12.

TABLE 12 Distribution of Products Including Various Deletion Peptides,According to HPLC Analysis^(a) for the Assembly of ACP via HODhat-HOAt-and HODhbt-Based Coupling Reagents^(b) Coupling Preact. Coupling ACP-2Ile -Ile -Ile Val Asn Unkwn^(d) Entry Method Solvent Equiv.^(c) (min)(min) (%) (%) (%) (%) (%) (%) (%) 1 HDATU DCM 1.5 7 1.5 47 7 9 16  12  3— 2 N-HATU DCM 1.5 7 1.5 21 5 7 12  6 23  — 3 HDTU DCM 1.5 7 1.5 4 13  513  2 3 — 4 HDATU DCM 4 7 3 86 — — 2 5 3 — 5 N-HATU DCM 4 7 3 78 — — 4 77 — 6 HDTU DCM 4 7 3 31 5 7 26  3 4 — 7 HDATU DCM 4 7 10 88 — — — 7 — —8 N-HATU DCM 4 7 10 81 — — — 3 3 — 9 HDATU DMF 1.5 0 1.5 68 3 10  17  21 — 10 N-HATU DMF 1.5 0 1.5 87 — 3 1 2 7 — 11 HDTU DMF 1.5 0 1.5 30 15 19  22  3 4 — 12 HDATU DMF 2 0 5 97 — 1 — 2 — — 13 N-HATU DMF 2 0 5 94 —1 1 2 2 — 14 HDTU DMF 2 0 5 81 2 8 7 1 1 — 15 HDATU DMF 1.5 7 1.5 60 1 77 1 5 16  16 N-HATU DMF 1.5 7 1.5 77 — 2 — 3 11  7 17 HDTU DMF 1.5 7 1.531 15  15  26  — 5 8 18 HDATU DMF 4 7 1.5 72 — 5 3 — 18  2 19 HDTU DMF 47 1.5 57 3 9 11  2 4 14  20 HDATU DMF 3 7 3 64 — 1 1 — 4 20  21 HDATUDMF 3 25 3 75 — 4 3 3 5 9 22 HDATU DMF 4 7 30 95 — 1 — 2 1 — 23 N-HATUDMF 4 7 30 86 4 — 2 1 1 — 24 HDTU DMF 4 7 30 62 2 2 — 1 2 28  25DIC-HODhat DMF 4 7 30 55 — — — 12  30  — 26 DIC-HODhbt DMF 4 7 30 29 9 —— — 3 5 27 Pfp^(e)-HODhat DMF 1.5^(f) 7 1.5 5 — — — — — — 28Pfp^(e)-HODhbt DMF 1.5^(g) 7 1.5 6 — — — — — — 29 Pfp^(e)-HODhat DMF3^(f) 7 30 90 — 1 1 7 1 — 30 Pfb^(e)-HODhbt DMF 3^(g) 7 30 87 — 3 3 6 1— ^(a)A reversed-phase C-18 column was used with elution by a lineargradient over 20 min of 0.1% TFA in MeCN and 0.1% aqueous TFA from 1:19to 1:1, flow rate 0.1 ml/min. ^(b)In all HDATU- or HODhat-mediatedsyntheses, a bright-yellow-to-orange-red color change was observedwithin 2–5 min during amino acid coupling, except for Ile and Val.^(c)Couplings were carried out in DMF or CH₂Cl₂ in the presence of 2equivalents of DIEA per equivalent of Fmoc-amino acid/coupling reagent.^(d)Extra peaks which appeared after 20 min on the HPLC traces.^(e)Pentafluorophenyl ester. ^(f)Only 1 equiv of HODhat was used ascatalyst. ^(g)Only 1 equiv of HODhbt was used as catalyst.

Analysis of the chromatograms indicated that HDATU is far more effectivethan HDTU under all conditions examined, and more effective even thanN-HATU in many instances. Methylene chloride was found to be aparticularly suitable solvent for HDATU-mediated ACP synthesis. Thus,under so called “1.5×1.5” conditions in CH₂Cl₂, HDATU gave thedecapeptide in a purity of 47%, whereas N-HATU and HDTU led to only 21%and 4% of the desired product, respectively. When a 4-equiv excess ofreagents and a 3-min coupling time were used, 86% of acyl carrierprotein (ACP) was obtained for HDATU, compared with 78% and 31% forN-HATU and HDTU, respectively.

In DMF under “1.5×1.5” conditions, the performance of HDATU may not havebeen as efficient as N-HATU with or without preactivation. In addition,for preparation of model pentapeptide H-Tyr-Aib-Aib-Phe-Leu-NH₂ (SEQ IDNO:3) which incorporates the highly hindered Aib-Aib unit, whether inDCM or DMF, HDATU was not able to equal the results obtained withN-HATU. For example, with 4 equivalents excess acid, 7 min preactivationand 30 min coupling time HDATU gave in DMF a peptide of 31% purity,whereas with N-HATU the purity was 91%.

However, in general HDATU was found to be the better reagent undernormal conditions. Thus, while using a 2-equiv excess of reagentswithout preactivation for a 5-min coupling, ACP was obtained in 97%purity by HDATU, whereas the corresponding values were 94 and 81% forN-HATU and HDTU. With 4-equiv/30 min coupling conditions with a 7-minpreactivation time, excellent purity (95%) was obtained for HDATU,whereas with N-HATU and HDTU, the ACP purity was only 86 and 62%,respectively.

When DIC/HODhat was used as a coupling reagent, satisfactory resultswere also obtained. Although not suitable under stringent conditions(“1.5×1.5”), HODhat could be used as an excellent catalyst and indicatorin Fmoc-amino acid pentafluorophenyl (Pfp) ester couplings under normalconditions. A bright-yellow-to-orange-red color change was noted whichis much clearer than the color change from bright-yellow to pale-yellowobserved with HODhbt. In DMF under conditions involving 3 equiv ofpentafluorophenyl-ester and a 30-mm coupling time, both HODhat andHODhbt gave the desired ACP product in a purity of over 85%.

EXAMPLE 27 Fmoc-Ile-ODhat

Method A. Under an atmosphere of dry N₂, a suspension of Fmoc-Ile-OH(0.3534 g, 1 mmol), HODhat (0.1805 g, 1.1 mmol) and SOCl₂ (0.73 ml, 10mmol) in 8 ml of dry CH₂Cl₂ was refluxed overnight. Evaporation ofCH₂Cl₂ and the excess of SOCl₂ gave a yellow solid, which was purifiedby flash chromatography through a short silica gel column with a mixtureof EtOAc—CH₂Cl₂ (1:1 v/v) as eluent to give, after tworecrystallizations from CH₂Cl₂-benzene-ether-hexane, 0.42 g (81%) of theanalytically pure ester as a white solid: mp 160.5-162° C.; ¹H NMR(CDCI₃): δ 9.15 (dd, 1), 8.58 (dd, 1), 7.96 (dd, 1), 7.76 (dd, 2), 7.61(dd, 2), 7.27-7.44 (m, 4), 5.20 (d, 1), 4.88 (q, 1), 4.49 (d, 2), 4.26(t, 1), 2.21 (m, 1), 1.70 (m, 1), 1.34 (m, 1), 1.15 (d, 3), 1.05 (t, 3);IR (KBr): 1811(s, COO), 1738(vs, CONN), 1692(vs, NHCO) cm⁻¹. Anal. Calcdfor C₂₇H₂₅N₅0₅: C, 64.91; H, 5.04; N, 14.02. Found: C, 64.77; H, 5.23;N, 13.94.

Method B. Under an atmosphere of dry N₂, 0.1854 g (0.5 mmol) ofFmoc-Ile-Cl was added with stirring to a solution of HODhat (0.0821 g,0.5 mmol) and DIEA (95.8 μl, 0.55 mmol) in 10 ml of CH₂Cl₂ at 0° C.Stirring was continued at 0° C. for 30 min and then at room temperaturefor 5 hours. The resulting light yellow solution was diluted to 30 mlwith CH₂Cl₂ and washed quickly with ice-cold brine (2×15 ml). Afterdrying over MgSO₄ and removing the solvent, the light yellow stickysolid was recrystallized twice from CH₂Cl₂-ether-hexane to give theanalytically pure above-identified ester as a white solid: mp 161-162°C.; NMR and IR spectra were identical with those of the sample obtainedby Method A.

EXAMPLE 28

Utilizing the procedure described in footnote a of Table 2 of thearticle by Carpino, et al., J. Org. Chem. 1995, 60, 3561, the contentsof which are incorporated by reference, the coupling of CBZ-Phe-Val-OHwith H Pro-NH₂ to form CBZ-Phe-Val-Pro-NH₂ was investigated usingvarious coupling reagents. Some of the coupling reagents used were thosedescribed elsewhere in the art while others used were coupling agents ofthe present invention. More specifically, for carbodiimide couplings,0.105 mmol of Z-Phe-Val-OH, 0.1 mmol of H-Pro-NH₂, and 0.11 mmol of thecoupling reagent noted hereinbelow in the table were dissolved in 1 mLof DMF or 1.3 mL of TFE/TCM (trifluoro ethanol/chloroform) (1:3), andthe solution was cooled in an ice bath and treated with 0.11 mmol ofEDC, EDC-HCl, or DCC. If a base is added, the number of equivalents isgiven. The mixture was stirred at 0° C. for 1 hour and at roomtemperature overnight. The mixture was diluted with 25 mL of EtOAc andextracted with 1 N NCl (2×5 mL), 1 N NaHCO₃ (2×5 mL), and saturated NaCl(2×5 mL), dried with MgSO₄, the solvent was removed, and the crudepeptide was directly analyzed by HPLC. For onium salt couplings, 0.125mmol of the acid, 0.125 mmol of amide, and 0.25 mmol of base in 1 mL ofDMF was treated with 0.125 mmol of coupling reagent at 0° C. and thereaction mixture was stirred at 0° C. for 1 hour and at room temperaturefor 2-3 hours, after which the workup followed that described herein. Incases where an additive is used, one or more equivalents of base (givenin parentheses) may be added. The amount of loss of configuration, asindicated by the presence of LDL epimer was determined. The results aretabulated in Table 13.

TABLE 13 [2 + 1] Segment coupling in solution (CBZ-Phe-Val-OH +H-Pro-NH₂). Coupling reagent Base Solvent % of LDL epimer O-HATUDCHMA^(b) (2 eq) DMF 7.91 t-Bu-Dtp-OAt DIEA (2eq) DMF 13.59 t-Bu-Dtp-OAtDCHMA^(b) (2 eq) DMF 15.81 O-HATU DIEA (2 eq) DMF 10.07 Dtp-OAt TMP(2eq) DMF 1.77 Dtp-OAt TMP (2eq)/HOAt (1 eq) DMF 1.9 t-Bu-Dtp-OAt TMP(2eq) DMF 1.63 O-HATU DB(DMAP) (2 eq) DMF 1.59 O-HATU Proton Sponge (2eq) DMF 2.78 Dtp-OAt DIEA (2 eq) DMF 16.04 O-HATU DIEA/TMP (1/1 eq) DMF10.17

EXAMPLE 29

Using the procedure as described in the article by Carpino, et al. inTetrahedron 1999, 55, 6813, the contents of which are incorporated byreference, the coupling of Fmoc-Asp (t-Bu)-Phe-OH and F-moc-Lys(BOC)-PAL-PEG to form Fmoc-Asp-(t-Bu)-Phe-Lys(-Boc)-PAL-PEG wasconducted. Some of the coupling reagents used were those describedelsewhere while others used were those of the present invention.Coupling reactions were carried out by deblocking 50 mg ofH-Lys(Boc)-PAL-PEG-PS resin by means of 20% piperidine/DMF for 7 min,washing the resin with DMF, DCM and DMF (3×5 mL each) and then adding a5-fold excess (0.0475 mmol) of Fmoc-Asp(O-t-Bu)-Phe-OH (26.5 mg), a5-fold excess of the coupling reagent noted in Table 14 and 11.5 mg(0.095 mmol) of TMP or 12.3 mg of DIEA (10-fold excess) of the base, ifany. In each case the coupling reagent and the base were dissolved in0.2 mL of the solvent and the resulting solution added to the resin in asmall syringe which served as the reactor. Dissolution required about 1min or less and care was taken to add the solution as soon as possibleafter everything dissolved. This method is referred to as the “lowpreactivation” method. Where preactivation was involved, the times arerecorded. The mixture was stirred gently every 10 min with a Teflon rodfor approximately 1 hour and then allowed to stand for 12 hours afterwhich the resin was washed with DMF and DCM (3×5 mL each) and deblockedby treatment with 3 mL of TFA/H₂O (9:1) for 1½ hour at room temperature.The solvent was removed in vacuo and the residue dissolved in CH₃—CN fordirect injection onto an HPLC column under the following conditions: 4μ60 A, C₁₈ Waters Nova-pak column, 3.9×150 mm; flow rate 1 mL/min; Waters996 PDA detector; linear gradient 10/30 in 20 min and then isocratic30/70 for 20 min with CH₃CN/H₂O/0.1% TFA; R_(t) (LLL-) 28.5 min, (LDL-)30.5 min. The amount of loss of configuration as indicated by thepresence of LDL epimer was determined. The results are tabulated inTable 14.

TABLE 14 [2 + 1] Segment coupling under solid phase conditions⁶(FmocAsp(tBu)-Phe-OH + H-Lys(BOC)-PAL-PEG). Coupling reagent BaseSolvent % of LDL epimer N-HATU TMP (2 eq) DMF 18.88 N-HATU TMP (2 eq)DCM 35.95 Dtp-OAt TMP (2 eq) DCM 14.70 t-Bu-Dtp-OAt TMP (2 eq) DCM 12.94Dtp-OAt TMP (2 eq) DMF 42.82

EXAMPLE 30

Utilizing the procedure of Example 29, and utilizing CBZ-Gly-Gly-Val-OHand H-Ala-Gly-Gly-PAL-Peg, CBZ-Gly-Gly-Val-Ala-Gly-Gly-PAL-PEG (SEQ IDNO:4) was formed using t-Bu-DtP-OAt of the present invention and O-HATU.The amount of loss of configuration was determined by measuring theamount of LDL epimer formed. The results are indicated in Table 15hereinbelow.

TABLE 15 [3 + 3] Segment coupling under solid phase conditions(CBZ-Gly-Gly-Val-OH + H-Ala-Gly-Gly-PAL-PEG). Coupling reagent BaseSolvent % of LDL epimer t-Bu-Dtp-OAt TMP DMF 1.99 O-HATU TMP DMF 2.09

The high coupling efficiency of the coupling reagents of the presentinvention including t-Bu-Dtp-OAt was emphasized by solid phase synthesisof ACP decapeptide under the so-called “1.5×1.5” protocol. Under thesedemanding conditions the coupling efficiency of various couplingreagents can be easily brought out. The couplings are carried out for1.5 minutes using a 1.5-eq excess of protected amino acids and 1.5 eq ofcoupling reagent in the presence of 3 eq of base.

For manual solid phase syntheses of ACP under the 1.5×1.5 protocol,using the procedure of Carpino, et al., in J. Chem. Soc. Chem. Comm,1994, 201, the contents of which are incorporated by reference and usingO-HATU, Dtp-OAt and t-Bu-Dtp-OAt the purity of the crude peptide was76%, 60% and 74%, respectively. The new phosphorus-based couplingreagent therefore at least equals the effectiveness of O-HATU,considered the best of the previously described reagents.

The above preferred embodiments and examples are given to illustrate thescope and spirit of the present invention. These embodiments andexamples will make apparent to those skilled in the art otherembodiments and examples. These other embodiments are within thecontemplation of the present invention. Therefore, the present inventionshould be limited only by the appended claims.

1. A compound or a salt consisting of an anion and, wherein the compoundor the cation of the salt is of the formula

wherein R₁ and R₂ taken together with the carbon atoms to which they areattached form a pyridyl ring, said pyridyl ring may be unsubstituted orsubstituted with a lower alkyl group or an electron donating group; Y₁is N; Q₁ is CR₁₆; R₁₆ is H or lower alkyl; R₁₄ is a positively chargedelectron withdrawing group,

SO₂R₁₇, lower alkyl carbonyl, lower arylalkyl carbonyl, aryl carbonyl,lower alkyl aryl, or BLK₁-AA₁ R₁₇ is aryl, aryl lower alkyl or loweralkyl; AA₁ is an amino acid or peptide less a hydrogen atom on theN-terminus and an OH on the C-terminus; BLK₁ is an amino protectinggroup, R₁₀ is OR₁₂, lower alkyl, aryl, aryl lower alkyl, lowercycloalkyl, lower cycloalkyl lower alkyl, heterocyclic, heterocycliclower alkyl, lower cycloalkenyl, or lower cycloalkenyl lower alkyl; R₁₁is OR₁₃, lower alkyl, aryl, aryl lower alkyl, lower cycloalkyl, lowercycloalkyl lower alkyl, heterocyclic, heterocyclic lower alkyl, lowercycloalkenyl or lower cycloalkenyl lower alkyl; and R₁₀ and R₁₁ mayoptionally be connected by a bridging group selected from the groupconsisting of O, S, NR₃₀, or (CHR₃₀)_(m), wherein each R₃₀ isindependently lower alkyl or hydrogen and m is 1-3; and R₁₂ and R₁₃ areindependently lower alkyl, lower cycloalkyl, lower cycloalkyl loweralkyl, heterocyclic, heterocyclic lower alkyl, lower cycloalkenyl orlower cyclalkenyl lower alkyl; ring A₁ and ring B are independently anaromatic ring containing 6 to 14 ring carbon atoms or cycloalkenyl orcycloalkyl, each containing 5 to 14 ring carbon atoms, and R_(b1),R_(c1), R_(b2), R_(c2) are independently hydrogen, lower alkyl orelectron donating group; T is CHR₃₁, O, S or NR₃₀; and R₃₁ is hydrogenor lower alkyl.
 2. The salt according to claim 1 wherein R₁₄ is apositively charged electron withdrawing group.
 3. The salt according toclaim 2 wherein R₁₄ is an electron withdrawing group of the formula

wherein R₁₈, R₁₉, R₂₀, R₂₁, R₂₂, R₂₃, R₂₄ and R₂₅ are independentlyhydrogen, lower alkyl, or lower alkoxy lower alkyl or R₁₈ and R₁₉ takentogether with the atoms to which they are attached form a ringcontaining up to 6 ring atoms and up to a total of 5 carbon ring atomsor R₂₀ and R₂₁ taken together with the nitrogen atom to which they areattached form a 5 or 6 membered nitrogen containing heterocyclic ringcontaining up to a total of 5 carbon ring atoms or R₁₈ and R₂₀ takentogether with the nitrogen atom and the carbon atom to which they areattached form a heterocyclic ring, or R₂₂ and R₂₃ taken together withthe atoms to which they are attached form a ring containing up to 6 ringatoms and up to a total of 5 carbon atoms or R₂₄ and R₂₅ taken togetherwith the carbon atoms to which they are attached form a ring containingup to 6 ring atoms and up to a total of 5 carbon atoms.
 4. The saltaccording to claim 3 wherein R₁₄ is

wherein R₁₉, R₂₁, R₂₄ and R₂₅ are independently hydrogen, or lower alkylor lower alkoxy lower alkyl; and n₁ is 0 or
 1. 5. The salt according toclaim 4 wherein R₁₉ and R₂₁ or R₂₄ and R₂₅ are the same.
 6. The saltaccording to claim 1 wherein R₁₄ is

wherein R₁₈, R₁₉, R₂₀, R₂₁, R₂₂, R₂₃, R₂₄ and R₂₅ are independentlyhydrogen, methyl, ethyl, propyl, butyl, pentyl, or CH₂CH₂OCH₂CH₃.
 7. Thesalt according to claim 6 wherein R₂₃, R₂₂, R₂₀, R₂₁, R₂₄, and R₂₅ arethe same or R₁₈, R₁₉ and R₂₀ are the same or R₁₉ and R₂₁ are the same.8. The compound or salt according to claim 1 wherein R₁₄ is

lower alkyl carbonyl, lower arylalkyl carbonyl, aryl carbonyl,

wherein U is NH, or O.
 9. The compound according to claim 1 wherein R₁₄is


10. A compound or a salt consisting of an anion and cation, wherein thecompound or the cation of the salt is of the formula

wherein R₁ and R₂ taken together with the carbon atoms to which they areattached form a pyridyl ring, said pyridyl ring may be unsubstituted orsubstituted with a lower alkyl group or an electron donating group; Y isN; Q₁ is CR₁₆; R₁₆ is H or lower alkyl; R₁₄ is

R₁₀ is OR₁₂, lower alkyl, aryl, or aryl lower alkyl; R₁₁ is OR₁₃, loweralkyl, aryl, or aryl lower alkyl; and R₁₀ and R₁₁ may optionally beconnected by a bridging group selected from the consisting of O, S, NH,and (CH₂)_(m); m is 1-3; and R₁₂ and R₁₃ are independently lower alkyl,aryl, or aryl lower alkyl.
 11. The compound according to claim 1 wherein

wherein R₁₀ and R₁₁ are independently lower alkyl or aryl.
 12. Thecompound according to claim 1 wherein

wherein R₁₁ and R₁₂ are independently lower alkyl or aryl.
 13. Thecompound or salt according to claim 1 wherein the compound or the cationof the salt has the formula

wherein R₁, R₂, Q₂ and R₁₄ are as defined in claim
 1. 14. A compound ora salt consisting of an anion and cation, wherein the compound or thecation of the salt has the formula

wherein A is N or CR₂₄; D is CR₂₅ or N; E is CR₂₆ or N; G is CR₂₇ or N;R₂₄, R₂₅, R₂₆ and R₂₇ are independently hydrogen, a lower alkyl group oran electron donating group, or R₂₅ and R₂₆ or R₂₄ and R₂₅ or R₂₆ and R₂₇taken together with the carbon atoms to which they are respectivelyattached form an aryl ring; wherein one of A, D, E and G, is N; Y₁ is N;Q₁ is CR₁₆; R₁₆ is H or lower alkyl; R₁₄ is a positively chargedelectron withdrawing group,

SO₂R₁₇, lower alkyl carbonyl, aryl carbonyl, lower alkyl aryl, orBLK₁-AA₁ R₁₇ is aryl, aryl lower alkyl or lower alkyl; AA₁ is an aminoacid or peptide less a hydrogen atom on the N-terminus and an OH on theC-terminus; BLK₁ is an amino protecting group, R₁₀ is OR₁₂, lower alkyl,aryl, aryl lower alkyl, lower cycloalkyl, lower cycloalkyl lower alkyl,heterocyclic, heterocyclic lower alkyl, lower cycloalkenyl, or lowercycloalkenyl lower alkyl; R₁₁ is OR₁₃, lower alkyl, aryl, aryl loweralkyl, lower cycloalkyl, lower cycloalkyl lower alkyl, heterocyclic,heterocyclic lower alkyl, lower cycloalkenyl or lower cycloalkenyl loweralkyl; and R₁₀ and R₁₁ may optionally be connected by a bridging groupselected from the group consisting of O, S, NR₃₀, or (CHR₃₀)_(m),wherein each R₃₀ is independently lower alkyl or hydrogen and m is 1-3;and R₁₂ and R₁₃ are independently lower alkyl, lower cycloalkyl, lowercycloalkyl lower alkyl, heterocyclic, heterocyclic lower alkyl, lowercycloalkenyl or lower cyclalkenyl lower alkyl; ring A₁ and ring B areindependently an aromatic ring containing 6 to 14 ring carbon atoms orcycloalkenyl or cycloalkyl, each containing 5 to 14 ring carbon atoms,and R_(b1), R_(c1), R_(b2), R_(c2) are independently hydrogen, loweralkyl or electron donating group; T is (CHR₃₁), O, S or NR₃₁; and R₃₁ ishydrogen or lower alkyl.
 15. The compound or salt according to claim 14where the compound or the cation has the formula

wherein A is N or CR₂₄; D is CR₂₅ or N; E is CR₂₆ or N; G is CR₂₇ or N;R₂₄, R₂₅, R₂₆ and R₂₇ are independently hydrogen or lower alkyl; whereinone of A, D, E and G, is N; Y₁ is N; Q₁ is CR₁₆; R₁₆ is H or loweralkyl; R₁₄ is a positively charged electron withdrawing group,

SO₂R₁₇, lower alkyl carbonyl, aryl carbonyl, loweralkyl aryl, orBLK₁-AA₁ R₁₇ is aryl, aryl lower alkyl or lower alkyl; AA₁ is an aminoacid or peptide less a hydrogen atom on the N-terminus and an OH on theC-terminus; BLK₁ is an amino protecting group, R₁₀ is OR₁₂, lower alkyl,aryl, aryl lower alkyl, lower cycloalkyl, lower cycloalkyl lower alkyl,heterocyclic, heterocyclic lower alkyl, lower cycloalkenyl, or lowercycloalkenyl lower alkyl; R₁₁ is OR₁₃, lower alkyl, aryl, aryl loweralkyl, lower cycloalkyl, lower cycloalkyl lower alkyl, heterocyclic,heterocyclic lower alkyl, lower cycloalkenyl or lower cycloalkenyl loweralkyl; and R₁₀ and R₁₁ may optionally be connected by a bridging groupselected from the group consisting of O, S, NR₃₀, or (CHR₃₀)_(m),wherein each R₃₀ is independently lower alkyl or hydrogen and m is 1-3;and R₁₂ and R₁₃ are independently lower alkyl, lower cycloalkyl, lowercycloalkyl lower alkyl, heterocyclic, heterocyclic lower alkyl, lowercycloalkenyl or lower cyclalkenyl lower alkyl; ring A₁ and ring B areindependently an aromatic ring containing 6 to 14 ring carbon atoms orcycloalkenyl or cycloalkyl, each containing 5 to 14 ring carbon atoms,and R_(b1), R_(c1), R_(b2), R_(c2) are independently hydrogen, loweralkyl or electron donating group; T is (CHR₃₁), O, S or NR₃₁; and R₃₁ ishydrogen or lower alkyl.
 16. The compound according to claim 15 whereinR₁₄ is

wherein R₁₀ and R₁₁, R_(b1), R_(b2), R_(c1), R_(c2) are independentlyhydrogen or lower alkyl and T is O, CH₂, NH or S and ring A₁ and ring Bare independently an aromatic ring containing 6 to 14 ring carbon atoms.17. The compound according to claim 1 wherein

wherein R₁₂, R₁₁, R_(b1), R_(b2), R_(c1) and R_(c2) are independentlyhydrogen or lower alkyl; ring A₁ and ring B are independently phenyl;and T is CH₂, O, S or NH.
 18. The compound according to claim 1, whereinthe compound is a salt, the cation of which has the formula


19. The compound according to claim 1 wherein the compound has theformula

wherein R_(b), R_(b1), R_(c), are independently lower alkyl or hydrogenand T is CH₂, NH, O or S.